Method for purifying active GLA-domain coagulation proteins

ABSTRACT

A a method for purifying biologically active GLA-domain coagulation proteins, includes the following steps: a) bringing a sample that contains one or more GLA-domain coagulation proteins and may contain biologically inactive molecules of GLA-domain protein(s), into contact with an affinity substrate on which nucleic aptamers that bind specifically to at least one biologically active GLA-domain coagulation protein are immobilized, in order to form complexes between (i) the nucleic aptamers and (ii) the GLA-domain coagulation protein(s), b) releasing the GLA-domain coagulation protein(s) from the complexes formed in step a), and c) recovering the biologically active GLA-domain coagulation protein(s) in a purified form.

FIELD OF THE INVENTION

The present invention relates to the field of protein purification, andin particular to the field of the purification of GLA-domain coagulationproteins, and even more specifically active GLA-domain coagulationproteins.

PRIOR ART

Generally, GLA-domain proteins form a family of proteins having a commonstructure, the GLA domain, which consists of a region located toward theN-terminal end of these proteins and which comprises a plurality ofglutamine residues which are in particular carboxylated to givecarboxyglutamic acid or “GLA” residues. GLA-domain proteins generallycomprise an N-terminal portion called a propeptide which is recognizedby a vitamin K-dependent carboxylase. After carboxylation of theglutamine residues of the GLA domain, the propeptide is cleaved byproteolysis and the mature and active GLA-domain protein is released.

Depending on the proteins under consideration, the GLA domain consistsof approximately 45 amino acids comprising from 9 to 12 glutamineresidues which are normally carboxylated to give Gla.

GLA-domain proteins consist of “vitamin K-dependent” proteins.GLA-domain proteins encompass coagulation factors, bone tissue proteinsand conopeptides. GLA-domain coagulation factors encompass prothrombin(Factor II), Factor VII, Factor IX, Factor X, protein C and protein S.GLA-domain bone tissue proteins encompass osteocalcin and matrix Glaprotein. GLA-domain conopeptides encompass conantokin G and conantokinT. GLA-domain proteins also encompass other proteins, such as protein Z,Gas6, PRGP1 and PRGP2. GLA-domain proteins are presented in particularin the article by Furie et al. (1999, Blood, Vol. 93: 1798-1808).

Vitamin K-dependent GLA-domain coagulation proteins consist of proteinsof therapeutic interest. Among said proteins, Factor II, Factor VII,Factor IX and Factor X represent proteins of very great therapeuticinterest which are administered for the prevention and treatment ofnumerous homeostasis disorders. Human GLA-domain coagulation proteinscan be purified from natural human fluids, generally from human bloodplasma. Moreover, numerous studies have been undertaken in order todevelop methods for producing and purifying recombinant human GLA-domaincoagulation proteins. Mention may be made, for example, of recombinanthuman Factor VII, which is already sold as a medicament.

It is understood that obtaining purified preparations in which thecoagulation factor(s) of interest is (are) in a biologically active formis of very great importance, since they are substances which are used asactive ingredients of medicaments.

Mention may in particular be made of the obtaining of purifiedpreparations of GLA-domain coagulation factors produced in the form ofrecombinant proteins, in vitro by means of genetically transformed cellsor in vivo by means of transgenic animals, for which it is essential toselect the biologically active forms of these recombinant factors whichare produced in artificial systems or in systems that are heterologousto humans.

For obtaining GLA-domain proteins purified from biological fluids inwhich these proteins are produced naturally or else in the form ofrecombinant proteins, suitable purification methods are already known inthe prior art. These methods generally comprise a succession ofselective separation steps based on steps of protein precipitation andof passage over chromatography supports, followed by sequential-elutionsteps, deep-filtration steps, ultrafiltration, or else concentrationsteps. The methods for purifying GLA-domain coagulation proteins thatare used today for producing medicaments do not comprise an affinitychromatography step. One reason for such a technical choice lies in thedrawbacks created by the detachment of a part of the ligand moleculesgrafted onto the affinity support, which are found associated with thepurified therapeutic protein in the volume of the chromatography eluate.By way of illustration, mention may be made of the product Mononine®,which is a pharmaceutical composition based on purified human Factor IX,which is obtained by means of a method using an immunoaffinity supporton which mouse anti-FIX monoclonal antibodies are immobilized. However,the monograph of the Mononine® product specifies the presence of tracesof murine anti-human FIX monoclonal antibodies in the final product,which is able to cause immunogenicity problems in treated patients sincethey become immunized against the “leachables” (murine antibodies andantibody fragments). The monograph of the Mononine® product specifiescontraindications for patients allergic to murine proteins.

It is known in the prior art that partial or total integrity of the GLAdomain of GLA-domain coagulation proteins, including prothrombin, FactorVII, Factor IX, Factor X, protein C and protein S, is important formaintaining their biological activity.

There therefore exists a need in the prior art for improved oralternative methods for purifying active GLA-domain coagulationproteins. This encompasses a need for alternative or improved methodsfor obtaining compositions comprising a single purified active protein,such as, for example, Factor VII or Factor IX, and also alternative orimproved methods for obtaining compositions comprising a combination ofactive GLA-domain proteins, for example compositions comprising acombination of active Factor II, active Factor VII, active Factor IX andactive Factor X. This encompasses a need for alternative or improvedmethods for purifying nonrecombinant active GLA-domain proteins orrecombinant active GLA-domain proteins.

SUMMARY OF THE INVENTION

The present invention relates to a method for purifying biologicallyactive GLA-domain coagulation proteins, comprising the following steps:

-   -   a) bringing a sample which contains one or more GLA-domain        coagulation proteins, and which may contain biologically        inactive molecules of GLA-domain protein(s), into contact with        an affinity support on which nucleic aptamers which bind        specifically to at least one biologically active GLA-domain        coagulation protein are immobilized, in order to form complexes        between (i) said nucleic aptamers and (ii) said active        GLA-domain coagulation protein(s),    -   b) releasing the active GLA-domain coagulation protein(s) from        the complexes formed in step a), and    -   c) recovering said biologically active GLA-domain coagulation        protein(s) in a purified form.

In the above methods, said aptamers are preferentially deoxyribonucleicaptamers.

In the above methods, said GLA-domain coagulation protein may be avitamin K-dependent coagulation factor, for example chosen from FactorII, Factor VII, Factor IX, Factor X, protein C and protein S.

In certain embodiments of these methods, said nucleic aptamers consistof aptamers of sequence SEQ ID NO. 4.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the curves of binding (i) of the Factor IX present invarious compositions to (ii) an aptamer specific for GLA-domain proteinswhich is immobilized on a support, in an assay according to the surfaceplasmon resonance technique. Along the x-axis: the time, expressed inseconds; along the y-axis, the resonance signal, expressed in arbitraryresonance units. Curve No. 1: recombinant FIX sold under the nameBeneFIX® by the company Wyeth; curve No. 2: semi-purified preparation ofrecombinant Factor IX produced by transgenic pigs (FIXtg). This FIX ispoorly gamma-carboxylated (carboxylation measurement <7 gammacarboxylations out of the 12 present); curve No. 3: negative control(buffer alone).

FIG. 2 is the image of an SDS-PAGE electrophoresis gel of proteinscontained in a purified fraction of human plasma Factor IX or milk froma pig transgenic for Factor IX, having undergone a pretreatment byclarification then chromatography on an MEP HyperCel® support, thenchromatography on an affinity support on which nucleic aptamers specificfor GLA-domain proteins are grafted. The SDS-PAGE gel was treated withCoomassie blue. From left to right: lane 1: starting composition: milkfrom a pig transgenic for human Factor IX, having undergone apretreatment by clarification then chromatography on an MEP HyperCel®support; lane 2: proteins contained in the fraction not retained on theaffinity support; lane 3: proteins contained in the elution fraction;lane 4: proteins contained, in the output from the affinity support, inthe regeneration buffer; lane 5: purified fraction of human plasmaFactor IX; lane 6: reference proteins of known molecular weight. Theapparent molecular weights of certain protein bands of the referenceproteins are indicated.

FIG. 3 is the image of an SDS-PAGE electrophoresis gel of proteinscontained in a purified fraction of human plasma Factor IX or in milkfrom a pig transgenic for human Factor IX, having undergone apretreatment by clarification then chromatography on an MEP HyperCel®support, then purification on an affinity support on which nucleicaptamers specific for GLA-domain proteins are grafted. The SDS-PAGE gelwas treated with silver nitrate. FIG. 4A represents the entire SDS-PAGEgel after staining. FIG. 4B is a detailed portion of the gel of FIG. 4A.Lanes “St” and “S”: reference proteins of known molecular weight; lanes1, 2 and 4: proteins contained in the elution fraction afterchromatography on an MEP HyperCel® support then purification on anaffinity support on which nucleic aptamers specific for GLA-domainproteins are grafted, following distinct assays; lane 3: purifiedfraction of human plasma Factor IX (25 ng of Factor IX loaded on theSDS-PAGE gel); lane 3′: purified fraction of human plasma Factor IX (550ng of Factor IX loaded on the SDS-PAGE gel). The apparent molecularweights of certain protein bands are indicated in FIGS. 4A and 4B.

FIG. 4 shows the curves of binding (i) the Factor IX present in variouscompositions to (ii) an aptamer specific for GLA-domain proteins whichis immobilized on a support, in an assay according to the surfaceplasmon resonance technique. Along the x-axis: the time, expressed inseconds; along the y-axis, the resonance signal, expressed in arbitraryresonance units. Curve No. 1: preparation of human plasma Factor IX;curve No. 2: preparation of recombinant Factor IX produced by atransgenic pig, purified by chromatography on an MEP HyperCel® supportthen on an affinity support comprising anti-GLA aptamers; curve No. 3:preparation of recombinant Factor IX produced by a transgenic pig,before running over an affinity support comprising anti-GLA aptamers(after MEP HyperCel®); curve No. 4: negative control preparation (bufferalone).

FIG. 5 shows the curves of binding (i) respectively of Factor VII, ofFactor IX or of Factor X, present in various compositions to (ii) anaptamer specific for GLA-domain proteins which is immobilized on asupport, in an assay according to the surface plasmon resonancetechnique. Along the x-axis: the time, expressed in seconds; along they-axis, the resonance signal, expressed in arbitrary resonance units.Curve No. 1: preparation of human recombinant Factor IX (BeneFIX®);curve No. 2: preparation of human plasma Factor VII; curve No. 3:preparation of human plasma Factor X; curve No. 4: negative controlpreparation.

FIG. 6 shows the curves of binding of a diversity of nucleic acids ofthe invention to recombinant human Factor IX which is immobilized on asupport, in an assay according to the surface plasmon resonancetechnique. Along the x-axis: the time, expressed in seconds; along they-axis, resonance signal, expressed in arbitrary resonance units. Curves“1”: low-affinity nucleic acids; curves “2”: intermediate-affinitynucleic acids; curves “3”: high-affinity nucleic acids; curves “4”: veryhigh-affinity nucleic acid.

FIG. 7 shows the curves of binding of the nucleic acid aptamers“Mapt-1.2.-CS” and “Mapt-1.2.-CSO” to recombinant human Factor IX whichis immobilized on a support, in an assay according to the surfaceplasmon resonance technique. Along the x-axis: the time, expressed inseconds; along the y-axis, resonance signal, expressed in arbitraryresonance units. Curve No. 1 is the curve of binding obtained with theMapt-1.2.-CS aptamer. Curve No. 2 is the curve of binding obtained withthe Mapt-1.2.-CSO aptamer.

FIG. 8 shows a chromatography profile obtained when carrying out themethod for purifying a human plasma Factor IX, produced with theaffinity support on which anti-GLA nucleic aptamers are immobilized.Along the x-axis: the time; along the y-axis: the absorbance value(O.D.) at 254 nanometers. (1): moment of injection of the human plasmaFIX concentrate; (2): nonretained fraction elimination peak; (3):purified FIX elution peak; (4): peaks generated during thechromatographic support regeneration step.

FIG. 9 is the image of an SDS-PAGE electrophoresis gel, with a gradientof from 4 to 12% of bisacrylamide without reducing agent, of proteinscontained in a purified fraction of human plasma Factor IX, havingundergone chromatography on an affinity support on which nucleicaptamers specific for GLA-domain proteins are grafted. The SDS-PAGE gelwas treated with Coomassie blue. From left to right: lane 1 (“SM”):starting composition, human plasma Factor IX concentrate; lane 2 (“NR”):proteins contained in the fraction not retained on the affinity support;lane 3 (“E1”): proteins contained in the elution fraction E1; lane 4(“E2”): proteins contained in the ouput from the affinity support, inthe regeneration buffer; lane 5 (“E3”): proteins contained in the outputfrom the affinity support, in the regeneration buffer; lane 6 (“T FIX”):purified fraction of human plasma Factor IX.

FIG. 10 shows a chromatography profile obtained when carrying out themethod for purifying a human plasma Factor IX, produced with theaffinity support in which anti-GLA nucleic aptamers are immobilized.Along the x-axis; the time; along the y-axis: the absorbance value(O.D.) at 254 nanometers. (1): nonretained fraction; (2): purified humanFIX elution peak; (3) chromatographic support regeneration peak.

FIG. 11 is the image of an SDS-PAGE electrophoresis gel, with a gradientof 4 to 12% of bisacrylamide without reducing agent, of proteinscontained in a purified fraction of human plasma Factor IX, havingundergone chromatography on an affinity support on which nucleicaptamers specific for GLA-domain proteins are grafted. The SDS-PAGE gelwas treated with Coomassie blue. From left to right: lane 1 (“Start”):starting composition, human plasma Factor IX concentrate; lane 2 (“Notretained”): proteins contained in the fraction not retained on theaffinity support; lane 3 (“Eluate”): proteins contained in the elutionfraction.

FIG. 12 shows a chromatography profile obtained when carrying out themethod for purifying a transgenic human Factor IX, produced in the milkof a transgenic sow, with the affinity support on which anti-GLA nucleicaptamers are immobilized. Along the x-axis: the time; along the y-axis:the absorbance value (O.D.) at 254 nanometers. (1): moment of injectionof the transgenic FIX, (2): nonretained fraction; (3): elution fraction.

FIG. 13 is the image of an SDS-PAGE electrophoresis gel of the proteinscontained in a prepurified fraction of recombinant human Factor IXproduced in the milk of a transgenic sow, having undergonechromatography on an affinity support on which nucleic aptamers specificfor GLA-domain proteins are grafted. The SDS-PAGE gel was treated withCoomassie blue. From left to right: lane 1 (“E5”, “Start”): startingcomposition, transgenic human Factor IX prepurified on MEP HyperCel®;lane 2 (“E6”, “Not Retained”): proteins contained in the fraction notretained on the affinity support; lane 3 (“E7”, “Elution”): proteinscontained in the elution fraction; lane 4 (“E8”, “Regeneration”):proteins contained in the regeneration fraction; lane 5 (“T FIX”, “PureFIX control”): purified FIX control. Arrow: position of migration ofFIX.

FIG. 14 shows the curves of binding of a diversity of nucleic acids ofthe invention to recombinant human Factor IX which is immobilized on asupport, in an assay according to the surface plasmon resonancetechnique. Along the x-axis: the time, expressed in seconds; along they-axis, resonance signal, expressed in arbitrary resonance units. Curves“1”: low-affinity nucleic acids; curves “2”: intermediate-affinitynucleic acids; curves “3”: high-affinity nucleic acids; curves “4”: veryhigh-affinity nucleic acid.

FIG. 15 shows a chromatography profile obtained when carrying out themethod for purifying a transgenic human Factor VII, produced with theaffinity support in which anti-GLA nucleic aptamers are immobilized.Along the x-axis; the time; along the y-axis: the absorbance value(O.D.) at 254 nanometers. (1): moment of injection; (2): nonretainedfraction; (3): moment of injection of the elution buffer; (4): elutionfraction.

FIG. 16 is the image of an SDS-PAGE electrophoresis gel of proteinscontained in a prepurified fraction of human plasma Factor VII, havingundergone chromatography on an affinity support on which nucleicaptamers specific for GLA-domain proteins are grafted. The SDS-PAGE gelwas treated with Coomassie blue. From left to right: (1): lane 1(“Start”): starting composition, human plasma Factor VII; (2): lane 2(“Eluate”): proteins contained in the elution fraction; (3): Des-GlaFVII: poorly glycosylated forms of FVII; (4): other poorly identifiedforms of FVII.

FIG. 17 shows a chromatography profile obtained when carrying out themethod for purifying a transgenic human factor VII, produced with theaffinity support in which anti-GLA nucleic aptamers are immobilized.Along the x-axis: the time; along the y-axis: the absorbance value(O.D.) at 254 nanometers. (1): nonretained fraction; (2): elutionfraction; (3): regeneration fraction.

FIG. 18 is the image of an SDS-PAGE electrophoresis gel of proteinscontained in a prepurified fraction of human plasma Factor VII, havingundergone chromatography on an affinity support on which nucleicaptamers specific for GLA-domain proteins are grafted. The SDS-PAGE gelwas treated with Coomassie blue. From left to right: (1): lane 1(“Start”): starting composition, human plasma Factor VII; (2): lane 2(“NR”): fraction of nonretained proteins; (3): lane 3: (“Eluate”):proteins contained in the elution fraction; (4): Des-Gla FVII, poorlyglycosylated forms of FVII.

FIG. 19 shows the curves of binding of the Mapt-2 aptamer which isimmobilized on a support, to human plasma Factor VII, in an assayaccording to the surface plasmon resonance technique. The curvescorrespond to the plasma FVII binding kinetics during the use of variousrunning buffers, respectively, from bottom to top of FIG. 16: T3: buffer3, T2: buffer 2, T1: buffer 1, T4: buffer 4 and T5: buffer 5. Along thex-axis: the time, expressed in seconds; along the y-axis: resonancesignal, expressed in arbitrary resonance units. From bottom to top ofthe figure: curves illustrating interactions of increasing affinity.

FIG. 20 shows the curves of binding of the Mapt-2 aptamer which isimmobilized on a support, to human plasma Factor VII, in an assayaccording to the surface plasmon resonance technique. The curve makes itpossible to determine the parameters of the plasma FVII binding kineticsduring the use of buffer 1 (50 mM Tris, 50 mM NaCl, 10 mM CaCl₂, 4 mMMgCl₂, pH 7.5). Along the x-axis: the time, expressed in seconds; alongthe y-axis, resonance signal, expressed in arbitrary resonance units.

FIG. 21 shows the curves of binding of the Mapt-2 aptamer which isimmobilized on a support, to human plasma Factor VII, in an assayaccording to the surface plasmon resonance technique. The curvecorresponds to the plasma FVII binding kinetics during the use of buffer5 (50 mM Tris, 10 mM CaCl₂, pH 7.5). Along the x-axis: the time,expressed in seconds; along the y-axis, resonance signal, expressed inarbitrary resonance units.

FIG. 22 shows the curves of binding of the Mapt-2 aptamer which isimmobilized on a support, to human plasma Factor VII, in an assayaccording to the surface plasmon resonance technique. The curvescorrespond to the resistance of the binding of plasma FVII to Mapt-2during the use of various washing buffers: (1): injection of FVII; (2):50 mM Tris buffer containing 1M NaCl, 10 mM CaCl₂, 4 mM MgCl₂, pH 7.5,(3): 50 mM Tris buffer containing 2M NaCl, 10 mM CaCl₂, 4 mM MgCl₂, pH7.5, (4): 50 mM Tris buffer containing 3M NaCl, 10 mM CaCl₂, 4 mM MgCl₂,pH 7.5 and (5): 50 mM Tris buffer containing 10 mM EDTA. Along thex-axis: the time, expressed in seconds; along the y-axis: resonancesignal, expressed in arbitrary resonance units.

FIG. 23 shows the curves of binding of the Mapt-2 aptamer which isimmobilized on a support, to human plasma Factor VII, in an assayaccording to the surface plasmon resonance technique. The curvescorrespond to the resistance of the binding of plasma FVII to Mapt-2when various washing buffers are used: (1): injection of FVII; (2): 50mM Tris buffer containing 10 mM CaCl₂, 4 mM MgCl₂, pH 7.5, (3): 50 mMTris buffer containing 10 mM CaCl₂, 4 mM MgCl₂, 1M NaCl, pH 7.5, (4): 50mM Tris buffer containing 10 mM CaCl₂, 4 mM MgCl₂, 2M NaCl, pH 7.5; (5):50 mM Tris buffer containing 10 mM CaCl₂, 4 mM MgCl₂, 3M NaCl, pH 7.5and (6) 50 mM Tris buffer containing 10 mM EDTA. Along the x-axis: thetime, expressed in seconds; along the y-axis, resonance signal,expressed in arbitrary resonance units.

FIG. 24 shows the curves of binding of the Mapt-2 aptamer which isimmobilized on a support, to human plasma Factor VII, in an assayaccording to the surface plasmon resonance technique. The curvescorrespond to the resistance of the binding of plasma FVII to Mapt-2when various washing buffers are used: (1): injection of FVII; (2): 10%ethanol buffer, (3): 50 mM Tris buffer containing 10 mM CaCl₂, 4 mMMgCl₂, 1M NaCl, pH 7.5, (4): 50 mM Tris buffer containing 10 mM CaCl₂, 4mM MgCl₂, 2M NaCl, pH 7.5; (5): 50 mM Tris buffer containing 10 mMCaCl₂, 4 mM MgCl₂, 3M NaCl, pH 7.5; and (6): 50 mM Tris buffercontaining 10 mM EDTA. Along the x-axis: the time, expressed in seconds;along the y-axis, resonance signal, expressed in arbitrary resonanceunits.

FIG. 25 shows the curves of binding of the Mapt-1 aptamer which isimmobilized on a support, to recombinant human Factor VII, in an assayaccording to the surface plasmon resonance technique. The curvescorrespond to the resistance of the binding of recombinant FIX to Mapt-1when various washing buffers are used: (1): injection of recombinantFVII; (2): 50 mM Tris buffer containing 10 mM CaCl₂, 4 mM MgCl₂, 1MNaCl, pH 7.5, (3): 50 mM Tris buffer containing 10 mM CaCl₂, 4 mM MgCl₂,2M NaCl, pH 7.5; (4): 50 mM Tris buffer containing 10 mM CaCl₂, 4 mMMgCl₂, 3M NaCl, pH 7.5 and (5): 50 mM Tris buffer containing 10 mM EDTA.Along the x-axis: the time, expressed in seconds; along the y-axis,resonance signal, expressed in arbitrary resonance units.

FIG. 26 shows the curves of binding of the Mapt-2 aptamer which isimmobilized on a support, to recombinant human Factor IX, in an assayaccording to the surface plasmon resonance technique. The curvescorrespond to the resistance of the binding of recombinant FIX to Mapt-1when the following washing buffer is used: 50 mM Tris, 10 mM CaCl₂, 50%propylene glycol, at pH 7.5. Along the x-axis: the time, expressed inseconds; along the y-axis, resonance signal, expressed in arbitraryresonance units. (1): injection of 50% propylene glycol in buffer 5.

DETAILED DESCRIPTION OF THE INVENTION

The applicant endeavors to design novel methods for purifying activeGLA-domain coagulation proteins, i.e. novel methods suitable forobtaining purified and active GLA-domain coagulation protein(s) from astarting sample comprising a GLA-domain coagulation protein or aplurality of GLA-domain coagulation proteins. In other words, theapplicant has sought to develop enrichment or purification methods whichare selective (i) for a given active GLA-domain coagulation protein, or(ii) for a plurality of active GLA-domain coagulation proteins.

In order to develop these purification methods, the applicant hasisolated and characterized novel ligands which have the ability to bindspecifically to an active GLA-domain protein or to a plurality of activeGLA-domain coagulation proteins.

These novel ligands do not detectably bind to a inactive GLA-domaincoagulation protein.

Furthermore, these novel ligands can be immobilized on the surface of asolid support in order to prepare supports for affinity purification ofGLA-domain coagulation proteins, said affinity purification supportshaving a reduced risk, or even a zero risk, of releasing the ligands andtherefore of contaminating the purified final product with said ligands,or with fragments or products resulting from the degradation of saidligands.

These novel ligands, the characteristics of which are detailed later inthe present description, consist of nucleic acids, also called “nucleicaptamers”, which bind to active GLA-domain coagulation proteins. Incertain embodiments, said aptamers are specific for a predeterminedactive GLA-domain coagulation protein. In other embodiments, saidaptamers are not specific for a particular active GLA-domain coagulationprotein and can bind to a plurality of active GLA-domain coagulationproteins.

The present invention provides methods for purifying active GLA-domaincoagulation proteins, in which advantage is taken of the bindingproperties of these novel ligands of the nucleic acid type.

The applicant has also endeavored to develop methods for obtainingnucleic aptamers which bind specifically to active GLA-domain proteins,with the objective of using said nucleic aptamers in methods forpurifying said active proteins.

According to the invention, nucleic aptamers which bind specifically toactive GLA-domain coagulation proteins have been obtained. Morespecifically, the applicant has obtained and characterized nucleicaptamers which bind exclusively to just one or to a plurality of activeGLA-domain coagulation protein(s), i.e. to proteins having a GLA domainof which the glutamine residues characteristic of the GLA domain havebeen at least partially gamma-carboxylated and of which the partialgamma-carboxylation of the GLA domain is sufficient for said GLA-domaincoagulation proteins to be active.

In certain embodiments, a nucleic aptamer of the invention, which isspecific for a predetermined GLA-domain coagulation protein, is capableof binding selectively to an active form of said GLA-domain coagulationprotein, and does not bind to an inactive form of said GLA-domainprotein.

In other embodiments, a nucleic aptamer of the invention, which isspecific for active GLA-domain proteins, is capable of binding withoutdistinction to a variety of proteins having a conserved commoncharacteristic which is the GLA domain, the gamma-carboxylation level ofwhich enables these proteins to be active, and does not bind to theinactive forms of said GLA-domain coagulation proteins.

It has in particular been shown in the examples that an aptamer specificfor GLA-domain coagulation proteins is capable of binding to a pluralityof active proteins, such as Factor IX, Factor VII and Factor X.

The results presented in the examples show that the anti-GLA aptamers ofthe invention, such as, for example, the Mapt-2-CS aptamer of sequenceSEQ ID NO. 37 or the Mapt-2.2.-CS aptamer of sequence SEQ ID NO. 38,bind exclusively to the active forms of GLA-domain coagulation proteins,in particular bind exclusively to the active forms of human Factor VII.For example, these anti-GLA aptamers do not bind to the Des-Gla forms ofhuman Factor VII, which consist of nonfunctional forms comprisingmodifications or an absence of the GLA domain.

Nucleic aptamers which specifically and individually recognizeGLA-domain proteins such as Factor II, Factor VII, Factor IX or Factor Xare already known in the prior art, including aptamers which bindthrombin (Zhao et al. 2008, Anal Chem, Vol 80(19): 7586-7593), aptamerswhich bind Factor IX/IXa (Subash et al., 2006, Thromb Haemost, Vol. 95:767-771; Howard et al., 2007, Atherioscl Thromb Vasc Biol, Vol. 27:722-727; PCT application No. WO 2002/096926; U.S. Pat. No. 7,312,325),aptamers which bind Factor X/Xa (PCT application No. WO 2002/096926;U.S. Pat. No. 7,312,325) or else aptamers which bind to human FactorVII/VIIa (Rusconi et al., 2000, Thromb Haemost, Vol. 84(5): 841-848;Layzer et al., 2007, Spring, Vol. 17: 1-11).

It is specified that none of the aptamers above is described for its usefor purifying the target protein to which it binds. Furthermore, theaptamers above bind exclusively to a single GLA-domain protein, withoutcrossing with another GLA-domain protein, thereby making it possible toimagine that the known aptamers do not bind to the GLA domain ofGLA-domain coagulation proteins, and are consequently not capable ofbinding selectively to the GLA domain of an active GLA-domain protein.

Such a type of aptamer, specific for a given GLA-domain protein, andwhich does not have the ability to bind to another protein, including toanother GLA-domain protein, is, for example, described by Layzer et al.(2007, Oligonucleotides, Vol. 17: 1-11).

However, to the applicant's knowledge, a nucleic aptamer, the bindingspecificity of which is the GLA domain of proteins, and which has theability to bind selectively to one or more active GLA-domain coagulationproteins, has never been described in the prior art.

Furthermore, to the applicant's knowledge, a nucleic aptamer, thebinding specificity of which is the GLA domain of proteins, and whichhas the ability to bind to a plurality of distinct active GLA-domaincoagulation proteins, has also not been described in the prior art.

The availability of nucleic aptamers which bind specifically to activeGLA-domain coagulation proteins has made it possible to develop methodsfor purifying these proteins, in particular with the objective ofobtaining a purified final product that can be used as an activeingredient of a medicament. Depending on the type of specificity of thenucleic aptamer which is used, said purification methods allow (i)either the selective purification of a predetermined active GLA-domaincoagulation protein, (ii) or the selective purification of the activeforms of a plurality of GLA-domain coagulation proteins.

The present invention relates to a method for purifying at least onebiologically active GLA-domain coagulation protein, comprising thefollowing steps:

-   -   a) bringing a sample which contains one or more GLA-domain        coagulation proteins into contact with an affinity support on        which nucleic aptamers which bind specifically to biologically        active GLA-domain coagulation proteins are immobilized, in order        to form complexes between (i) said nucleic aptamers and (ii)        said active GLA-domain coagulation protein(s),    -   b) releasing the biologically active GLA-domain coagulation        protein(s) from the complexes formed in step a), and    -   c) recovering said biologically active GLA-domain coagulation        protein(s) in a purified form.

The present invention also relates to a method for purifyingbiologically active GLA-domain coagulation proteins, comprising thefollowing steps:

-   -   a) bringing a sample which contains one or more GLA-domain        coagulation proteins into contact with an affinity support on        which nucleic aptamers which bind specifically to a plurality of        biologically active GLA-domain coagulation proteins are        immobilized, in order to form complexes between (i) said nucleic        aptamers and (ii) said GLA-domain coagulation protein(s),    -   b) releasing the biologically active GLA-domain coagulation        protein(s) from the complexes formed in step a), and    -   c) recovering said biologically active GLA-domain coagulation        protein(s) in a purified form.

The term “GLA-domain coagulation protein” encompasses any coagulationprotein comprising a region, denoted GLA domain, comprising a pluralityof glutamine residues which are gamma-carboxylated during proteinsynthesis. The GLA-domain coagulation proteins encompass coagulationproteins which have a GLA domain toward the N-terminal end, said GLAdomain generally being located downstream, i.e. on the C-terminal side,of a propeptide which is naturally hydrolyzed during synthesis. TheGLA-domain coagulation proteins encompass coagulation proteins in whichthe GLA domain consists of a region of approximately 45 amino acidscomprising from 9 to 12 glutamine residues, at least a part of which arenormally carboxylated to give GLA residues during the protein synthesisprocess in cells. The GLA-domain coagulation factors, which consist ofvitamin K-dependent proteins, encompass prothrombin (Factor II), FactorVII, Factor IX, Factor X, protein C and protein S. The GLA-domaincoagulation proteins encompass nonrecombinant proteins originating fromnatural sources, such as blood plasma, and also recombinant proteinswhich can be produced in vitro by cells transfected or transformed witha DNA encoding said coagulation protein or which can be produced in vivoby animals into which a transgene encoding said coagulation protein hasbeen introduced. The GLA-domain coagulation proteins produced bytransgenic animals may also be called “transgenic proteins” in thepresent description.

Methods for determining the activity of GLA-domain coagulation proteinsare described in detail later in the present description.

An “active” or “biologically active” GLA-domain coagulant proteinencompasses GLA-domain coagulation proteins which have at least half thelevel of anticoagulant or amidolytic activity that is determined for acorresponding reference protein of which respectively the anticoagulantactivity or the amidolytic activity is judged to be optimum. An activeGLA-domain coagulation protein encompasses GLA-domain coagulationproteins which have at least 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85,0.9, 0.95, 0.96, 0.97, 0.98, 0.99 or 1 times the level of anticoagulantor amidolytic activity that is determined for a corresponding referenceprotein of which respectively the anticoagulant activity or theamidolytic activity is judged to be optimum. An active GLA-domainprotein also encompasses GLA-domain proteins, which can also be denoted“superactive” GLA proteins, which have a level of anticoagulant oramidolytic activity greater than the reference protein or composition,i.e. which have a level of anticoagulant or amidolytic activity greaterthan 1.

Active Factor II (prothrombin) encompasses Factors II which have atleast half the level of anticoagulant or amidolytic activity that isdetermined for the corresponding reference Factor II of whichrespectively the anticoagulant activity or the amidolytic activity isjudged to be optimum.

Active Factor VII encompasses Factors VII which have at least half thelevel of anticoagulant or amidolytic activity that is determined for thecorresponding reference Factor VII of which respectively theanticoagulant activity or the amidolytic activity is judged to beoptimum.

Active Factor IX encompasses Factors IX which have at least half thelevel of anticoagulant or amidolytic activity that is determined for thecorresponding reference Factor IX of which respectively theanticoagulant activity or the amidolytic activity is judged to beoptimum.

Active Factor X encompasses Factors X which have at least half the levelof anticoagulant or amidolytic activity that is determined for thecorresponding reference Factor X of which respectively the anticoagulantactivity or the amidolytic activity is judged to be optimum.

Active protein C encompasses proteins C which have at least half thelevel of anticoagulant or amidolytic activity that is determined for thecorresponding reference protein C of which respectively theanticoagulant activity or the amidolytic activity is judged to beoptimum.

Active protein S encompasses proteins S which have at least half thelevel of anticoagulant or amidolytic activity that is determined for thecorresponding reference protein S of which respectively theanticoagulant activity or the amidolytic activity is judged to beoptimum.

In the light of the aforementioned, those skilled in the art understandthat an “active” or “biologically active” GLA-domain coagulation proteinshould not be confused with GLA-domain coagulation proteins which are“activated”. GLA-domain coagulation proteins are “activated” as a resultof a proteolysis reaction which cleaves a peptide, the resulting proteinthen exerting its own biological activity. The “activated” coagulationfactors are generally denoted with an additional “a”. By way ofillustration, the Factor VII/“activated” Factor VII pair can be denotedas the FVII/FVIIa pair. However, for the purpose of the invention, thereare both “biologically active” forms and “biologically inactive” formsof an “activated” coagulation factor, owing to the fact that there areforms of an “activated” GLA-domain coagulation factor which are“biologically inactive”, for example owing to an incorrectgamma-carboxylation of their GLA domain. Thus, for the purpose of theinvention, a “biologically active” GLA-domain coagulation proteinencompasses both (i) the “activated” form of said GLA-domain coagulationprotein, which directly exerts its own biological activity, and (ii) thenonactivated form of said protein, which nonactivated form of saidprotein results, after activation, in a form of said protein whichexerts its own biological activity.

The term “corresponding protein” or “corresponding factor” is intendedto mean preferentially the protein or the factor of the same mammal, forexample human being.

The active GLA-domain coagulation proteins, also called biologicallyactive GLA-domain coagulation proteins, encompass the proteins of thistype in which all of the glutamine residues of the GLA domain aregamma-carboxylated.

In certain cases, the active GLA-domain coagulation proteins encompassthe proteins of this type in which at least one glutamine residue of theGLA domain is not gamma-carboxylated, but which is neverthelessbiologically active for the purpose of the invention.

According to the invention, the term “nucleic aptamer” is intended tomean a single-stranded nucleic acid which binds specifically to one ormore active GLA-domain coagulation proteins, which may also be denotedin the present description as “anti-GLA aptamer”. The aptamers of theinvention therefore encompass those for which it is possible to detectcomplexes with a single active GLA-domain coagulation protein or with avariety of given active GLA-domain coagulation proteins, after a priorstep of bringing the respectively nucleic and protein partners intocontact. Preferentially, a “nucleic aptamer” according to the inventionhas the ability to bind to a plurality of active GLA-domain proteins.

The detection of complexes formed between an anti-GLA aptamer accordingto the invention and an active GLA-domain coagulation protein can beeasily carried out by those skilled in the art, for example byimplementing a surface plasmon resonance detection technique, includingthe Biacore® technique, as is illustrated in the examples. Those skilledin the art can also easily detect the formation of complexes between ananti-GLA aptamer according to the invention and an active GLA-domaincoagulation protein by conventional techniques of the ELISA type, as isknown by those skilled in the art.

It has been shown in the examples that an anti-GLA aptamer according tothe invention is capable of binding selectively to the active forms ofGLA-domain coagulation proteins, such as Factor IX. It has also beenshown according to the invention that an anti-GLA aptamer is capable ofbinding respectively to a plurality of distinct active GLA-domaincoagulation proteins, and in particular to a plurality of active humanGLA-domain coagulation proteins. It has in particular been shown that agiven anti-GLA aptamer according to the invention is capable of bindingrespectively to active Factor VII, to active Factor IX and to activeFactor X.

In certain embodiments, the purification method above is characterizedin that said GLA-domain coagulation protein is a vitamin K-dependentcoagulation factor.

In certain embodiments, the purification method above is characterizedin that said GLA-domain coagulation protein is chosen from Factor II,Factor VII, Factor IX, Factor X, protein C and protein S.

In certain embodiments, the purification method above is suitable forselectively purifying a single active GLA-domain coagulation proteinchosen from Factor II, Factor VII, Factor IX, Factor X, protein C andprotein S.

In certain embodiments, the purification method above is suitable forsimultaneously purifying at least two active GLA-domain coagulationproteins chosen from Factor II, Factor VII, Factor IX, Factor X, proteinC and protein S.

In certain embodiments, the purification method above is suitable forsimultaneously purifying at least three active GLA-domain coagulationproteins chosen from Factor II, Factor VII, Factor IX, Factor X, proteinC and protein S.

In certain embodiments, the purification method above is suitable forsimultaneously purifying active Factor II, active Factor VII, activeFactor IX and active Factor X.

In certain embodiments, the purification method above is suitable forpurifying active Factor IX.

It has thus been shown in the examples that an anti-GLA aptameraccording to the invention is capable of distinguishing activeGLA-domain coagulation proteins from inactive GLA-domain coagulationproteins. In particular, it has been shown in the examples that ananti-GLA aptamer according to the invention is capable of distinguishingGLA-domain coagulation proteins of which the GLA domain is correctlygamma-carboxylated from GLA-domain coagulation proteins of which the GLAdomain is incorrectly gamma-carboxylated.

According to the invention, a correctly gamma-carboxylated GLA domainencompasses a GLA domain of which all the characteristic glutamineresidues are gamma-carboxylated and also a GLA domain of which only apart of the characteristic glutamine residues are gamma-carboxylated,but of which the partial gamma-carboxylation does not lead to theGLA-domain coagulation protein under consideration not beingbiologically active, for the purpose of the invention.

By way of illustration, for a GLA-domain coagulation protein such asFactor IX, the protein is biologically active when all of thecharacteristic glutamine residues of its GLA domain aregamma-carboxylated, and also when at least 7 glutamine residues of theGLA domain are gamma-carboxylated, among the 12 characteristic glutamineresidues contained in its GLA domain. Thus, for Factor IX, the activeproteins encompass the Factors IX in which 7, 8, 9, 10, 11 and 12glutamine residues of the GLA domain have been gamma-carboxylated.

With regard to Factor IX, an anti-GLA nucleic aptamer according to theinvention encompasses the aptamers which bind to Factors IX in which 7,8, 9, 10, 11 and glutamine residues of the GLA domain have beengamma-carboxylated and which do not bind to Factors IX in which lessthan 7 glutamine residues of the GLA domain have beengamma-carboxylated. As an example of such an aptamer, mention may bemade of the aptamer of sequence SEQ ID NO. 4.

These properties of an anti-GLA aptamer of the invention, ofdistinguishing between active GLA-domain coagulation proteins andinactive GLA-domain coagulation proteins, can be used in methods forpurifying active GLA-domain coagulation proteins, in order to obtain afinal product which is greatly enriched in active form(s) of theGLA-domain coagulation protein(s) of interest. Examples ofimplementation of methods for purifying active forms of activeGLA-domain coagulation proteins, in particular active forms of FactorIX, are illustrated in the examples. These advantageous distinctionproperties of the anti-GLA aptamers of the invention make it possible,for example, to obtain purified preparations of active recombinant humanFactor IX from the milk of a mammal transgenic for human Factor IX, whensaid milk comprises both active forms and inactive forms of recombinanthuman Factor IX, in particular forms of recombinant human Factor IX ofwhich the GLA domain is correctly gamma-carboxylated and forms ofrecombinant human Factor IX of which the GLA domain is incorrectlygamma-carboxylated.

The present invention also relates to a method for purifyingbiologically active GLA-domain coagulation proteins, comprising thefollowing steps:

-   -   a) bringing a sample which contains one or more GLA-domain        coagulation proteins, and which may contain biologically        inactive molecules of GLA-domain coagulation protein(s), into        contact with an affinity support on which nucleic aptamers which        bind specifically to a biologically active GLA-domain        coagulation protein or to a plurality of biologically active        GLA-domain coagulation proteins are immobilized, in order to        form complexes between (i) said nucleic aptamers and (ii) said        active GLA-domain coagulation protein(s),    -   b) releasing the active GLA-domain coagulation protein(s) from        the complexes formed in step a), and    -   c) recovering said biologically active GLA-domain coagulation        protein(s) in a purified form.

In certain embodiments of the method above, said nucleic aptamersconsist of deoxyribonucleic aptamers.

In certain embodiments, the purification method above is characterizedin that said GLA-domain coagulation protein is a vitamin K-dependentcoagulation factor.

In certain embodiments, the purification method above is characterizedin that said GLA-domain coagulation protein is chosen from Factor II,Factor VII, Factor IX, Factor X, protein C and protein S.

In certain embodiments, the purification method above is suitable forselectively purifying a single active GLA-domain coagulation proteinchosen from Factor II, Factor VII, Factor IX, Factor X, protein C andprotein S.

In certain embodiments, the purification method above is suitable forsimultaneously purifying at least two active GLA-domain coagulationproteins chosen from Factor II, Factor VII, Factor IX, Factor X, proteinC and protein S.

In certain embodiments, the purification method above is suitable forsimultaneously purifying at least three active GLA-domain coagulationproteins chosen from Factor II, Factor VII, Factor IX, Factor X, proteinC and protein S.

In certain embodiments, the purification method above is suitable forsimultaneously purifying active Factor II, active Factor VII, activeFactor IX and active Factor X.

In certain embodiments, the purification method above is suitable forpurifying active Factor IX.

As has already been mentioned previously, an “active” form of aGLA-domain coagulation protein consists of a form for which the GLAdomain is correctly gamma-carboxylated, i.e. a form of the GLA-domaincoagulation protein which is recognized by an anti-GLA aptamer accordingto the invention.

An anti-GLA aptamer can be obtained by means of methods which have beenspecially developed for the needs of the invention, and which aredetailed later in the present description.

The simultaneous purification of more than one GLA-domain coagulationprotein depends in particular on the type of starting sample that isused to carry out the purification method. In particular, the number andthe identity of the GLA-domain coagulation proteins which are obtainedin purified form at the end of the method are logically limited by thenumber and the identity of the GLA-domain coagulation proteins which areinitially present in the starting sample.

For the purpose of the invention, the “purification” of an activeGLA-domain coagulation protein means an enrichment in the active form(s)of said GLA-domain coagulation protein, i.e. the obtaining of a“purified” composition having a concentration of the active GLA-domainprotein that is detectably greater than the initial concentration ofsaid active GLA-domain coagulation protein in the starting sample whichis subjected to the purification step. An active GLA-domain coagulationprotein “in purified form” encompasses compositions comprising an activeGLA-domain coagulation protein of which the concentration is detectablygreater than its concentration in the starting composition beforecarrying out the purification step. Thus, an active GLA-domaincoagulation protein of the invention encompasses a composition in whichsaid active GLA-domain coagulation protein is purified to homogeneity,but is in no way restricted to this particular purified form. Inparticular, when the sample before purification comprises more than oneGLA-domain coagulation protein, the purified active proteins cannot bydefinition be purified to homogeneity, simply because at least twoactive GLA-domain coagulation proteins coexist in the composition afterpurification.

Generally, the anti-GLA aptamers can consist of DNA (deoxyribonucleicacid) or RNA (ribonucleic acid) molecules and have an ability to bind toan active GLA-domain protein, which is greater than the ability to bindto an inactive GLA-domain coagulation protein.

An anti-GLA aptamer can be obtained by means of methods which have beenspecially developed for the needs of the invention, and which aredetailed later in the present description.

In certain embodiments, the anti-GLA aptamers according to the inventionhave various common structural characteristics, including a sequencecomprising, from the 5′ end to the 3′ end, successively (i) aninvariable specific nucleotide sequence of approximately 20 nucleotidesin length (for example 18 nucleotides in length), followed by (ii) avariable nucleotide sequence of approximately 40 to 50 nucleotides inlength (for example 44 nucleotides in length), followed by (iii) aninvariable specific nucleotide sequence of approximately 20 nucleotidesin length (for example 18 nucleotides in length). It is specified thatthe variable nucleotide sequences (ii) can have a very strong nucleotidesequence identity with respect to one another.

Some of the methods for selecting anti-GLA aptamers which are specifiedin the present description are of the type which makes it possible toobtain a family of anti-GLA aptamers, capable of selectively recognizinga predetermined active GLA-domain coagulation protein, in particular apredetermined human active GLA-domain coagulation protein.

Some of the other methods for selecting anti-GLA aptamers which arespecified in the present description are of the type which makes itpossible to obtain a family of anti-GLA aptamers, capable of selectivelyrecognizing a plurality of active GLA-domain coagulation proteins, inparticular a plurality of human active GLA-domain coagulation proteins.

From a structural point of view, the family of nucleic acids, or nucleicaptamers, which bind specifically to the active GLA-domain coagulationproteins of the invention comprises at least 15 consecutive nucleotidesof a polynucleotide having at least 40% nucleotide identity with thenucleic acid of formula (I) below:5′-[SEQ ID NO.1]x-[SEQ ID NO.X]-[SEQ ID NO.2]y-3′  (I),in which:

-   -   “SEQ ID NO. X” consists of a nucleic acid of sequence SEQ ID NO.        3,    -   “X” is an integer equal to 0 or 1, and    -   “Y” is an integer equal to 0 or 1.

In certain embodiments, the acid of sequence SEQ ID NO. X has a lengthof 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49or 50 nucleotides.

In other embodiments, the nucleic acid of sequence SEQ ID NO. X has alength of 43, 44, 45, 46, 47, 48 or 49 nucleotides.

In certain other preferred embodiments, the nucleic acid of sequence SEQID NO. X has a length of 43, 44 or 45 nucleotides.

As already mentioned previously, the nucleic acid of formula (I) is atleast 15 nucleotides in length.

In certain embodiments, the nucleic acid of formula (I) is at least 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80 or 81 nucleotides in length,thereby encompassing nucleic acids having exactly each of the lengthsspecified.

When the integer “x” is equal to 0 and the integer “y” is equal to 1,the nucleic aptamers of the invention encompass nucleic acids comprisingat least 15 consecutive nucleotides of a polynucleotide having at least40% nucleotide identity with the nucleic acid of formula (I-1) below:5′-[SEQ ID NO.X]-[SEQ ID NO.2]-3′  (I-1)

When the integer “x” is equal to 1 and the integer “y” is equal to 0,the nucleic aptamers of the invention encompass nucleic acids comprisingat least 15 consecutive nucleotides of a polynucleotide having at least40% nucleotide identity with the nucleic acid of formula (I-2) below:5′-[SEQ ID NO.1]-[SEQ ID NO.X]-3′  (I-2)

When the integer “x” is equal to 0 and the integer “y” is equal to 0,the nucleic aptamers of the invention encompass nucleic acids comprisingat least 15 consecutive nucleotides of a polynucleotide having at least40% nucleotide identity with the nucleic acid of formula (I-3) below:5′-[SEQ ID NO.X]-3′  (I-3)

The nucleic aptamers above therefore encompass nucleic acids comprisingat least 15 consecutive nucleotides of a polynucleotide having at least40% nucleotide identity with a nucleic acid of sequence SEQ ID NO. 3.

Generally, a first polynucleotide having at least 40% nucleotideidentity with a second polynucleotide or nucleic acid has at least 41%,42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%,56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 100% nucleotide identity with said second polynucleotide ornucleic acid.

In certain embodiments of a nucleic acid of the invention comprising thesequence SEQ ID NO. X, said sequence SEQ ID NO. X is chosen from thegroup consisting of nucleic acids having at least 15 consecutivenucleotides of a sequence having at least 40% nucleotide identity with asequence chosen from the sequences SEQ ID NOS. 3, 6 to 35, 37 and 38.

In certain embodiments of a nucleic acid of the invention comprising thesequence SEQ ID NO. X, said sequence SEQ ID NO. X is chosen from thegroup consisting of nucleic acids having at least 41%, 42%, 43%, 44%,45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%,59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100%nucleotide identity with a sequence chosen from the sequences SEQ IDNOS. 3, 6 to 35, 37 and 38.

In certain embodiments of a nucleic aptamer of the invention, saidaptamer is chosen from nucleic acids comprising a sequence of at least15 consecutive nucleotides of a sequence having at least 40% nucleotideidentity with a sequence chosen from the sequences SEQ ID NOS. 3, 4 and6 to 39.

In certain embodiments of a nucleic aptamer of the invention, saidaptamer is chosen from nucleic acids comprising a sequence of at least15 consecutive nucleotides of a sequence chosen from the sequences SEQID NOS. 3, 4 and 6 to 39.

In certain embodiments of a nucleic aptamer of the invention, saidaptamer is chosen from nucleic acids comprising a sequence having atleast 40% nucleotide identity with a sequence chosen from the sequencesSEQ ID NOS. 3, 4 and 6 to 39.

In certain embodiments of a nucleic aptamer of the invention, saidaptamer is chosen from nucleic acids comprising a sequence chosen fromthe sequences SEQ ID NOS. 3, 4 and 6 to 39.

In certain embodiments of a nucleic aptamer of the invention, saidaptamer is chosen from nucleic acids consisting of a sequence chosenfrom the sequences SEQ ID NOS. 3, 4 and 6 to 39.

It results from the aforementioned that the present inventionencompasses a family of single-stranded nucleic acids having at least 15consecutive nucleotides of the series of formula (I) defined above.

For the purpose of the present invention, the “percentage identity”between two nucleic acid sequences is determined by comparing the twooptimally aligned sequences, through a comparison window.

The part of the nucleotide sequence in the comparison window can thuscomprise additions or deletions (for example gaps) compared with thereference sequence (which does not comprise these additions or thesedeletions) so as to obtain an optimum alignment between the twosequences.

The percentage identity is calculated by determining the number ofpositions at which an identical nucleic base is observed for the twosequences compared, then by dividing the number of positions at whichthere is identity between the two nucleic bases by the total number ofpositions in the comparison window, and then by multiplying the resultby one hundred in order to obtain the percentage nucleotide identity ofthe two sequences with respect to one another.

The optimum alignment of the sequences for the comparison can be carriedout by computer using known algorithms.

Entirely preferably, the percentage sequence identity is determinedusing the CLUSTAL W software (version 1.82), the parameters being fixedas follows: (1) CPU MODE=ClustalW mp; (2) ALIGNMENT=“full”; (3) OUTPUTFORMAT=“aln w/numbers”; (4) OUTPUT ORDER=“aligned”; (5) COLORALIGNMENT=“no”; (6) KTUP (word size)=“default”; (7) WINDOWLENGTH=“default”; (8) SCORE TYPE=“percent”; (9) TOPDIAG=“default”; (10)PAIRGAP=“default”; (11) PHYLOGENETIC TREE/TREE TYPE=“none”; (12)MATRIX=“default”; (13) GAP OPEN=“default”; (14) END GAPS=“default”; (15)GAP EXTENSION=“default”; (16) GAP DISTANCES=“default”; (17) TREETYPE=“cladogram” and (18) TREE GRAPH DISTANCES=“hide”.

In certain preferred embodiments of an anti-GLA nucleic aptamer of theinvention, said nucleic aptamer comprises at least 15 consecutivenucleotides of a polynucleotide having at least 80% nucleotide identitywith the nucleic acid of formula (I), thereby encompassing the aptamerscomprising 15 consecutive nucleotides of a polynucleotide having atleast 81%, 820, 830, 840, 85%, 86%, 87%, 88%, 89%, 90%, 910, 920, 93%,94%, 95%, 96%, 97%, 98% or 100% nucleotide identity with said nucleicacid of formula (I).

The nucleic aptamers according to the invention encompass the nucleicaptamer of sequence SEQ ID NO. 4. It is recalled that the aptamer ofsequence SEQ ID NO. 4 consists of an aptamer of formula (I) in which thesequence SEQ ID NO. X consists of the sequence SEQ ID NO. 3. It is alsorecalled that an aptamer of sequence SEQ ID NO. 3 consists of an aptamerof formula (I-3) in which the sequences SEQ ID NO. 1 and SEQ ID NO. 2are absent.

The applicant has shown that the aptamer of sequence SEQ ID NO. 3 has anability to selectively bind to active GLA-domain coagulation proteinsthat is substantially identical to that of the aptamer of sequence SEQID NO. 4. These results show the essential nature of the presence of thenucleic acid of sequence SEQ ID NO. 3 in the specific binding propertiesof the aptamer of sequence SEQ ID NO. 4. Generally, these results showthe essential nature of the nucleic acid of sequence SEQ ID NO. X in anaptamer of formula (I), the nucleic acid of sequence SEQ ID NO. Xconferring on the aptamer of formula (I) the ability to bind selectivelyto the active GLA-domain coagulation proteins.

The nucleic aptamers of the invention therefore also encompass theaptamer of sequence SEQ ID NO. 3.

It has also been shown in the examples that a great variety of aptamers,including the aptamers of sequences SEQ ID NOS. 6 to 35, have an abilityto bind selectively to active GLA-domain coagulation proteins, whereappropriate with distinct affinity levels. By way of illustration, amongthe aptamers of sequences SEQ ID NOS. 6 to 35, the aptamer which has thegreatest ability to bind to human Factor IX is the aptamer of sequenceSEQ ID NO. 35, which can also be denoted “Mapt-1.2.-CS”.

Aptamers having an ability to bind to active GLA-domain coagulationproteins that is even greater than the Mapt-1.2.-CS aptamer above havealso been identified in the examples. An example of such an aptamer isthe aptamer of sequence SEQ ID NO. 36, which can also be denoted“Mapt-1.2.-CSO”.

The Mapt-1.2.-CSO aptamer of sequence SEQ ID NO. 36 is an aptamercomprising at least 15 consecutive nucleotides of a polynucleotidehaving at least 40% nucleotide identity with the nucleic acid of 62nucleotides in length, of formula (I) below:5′-[SEQ ID NO.1]-[SEQ ID NO.35]-3′, andconsisting of the nucleic acid which goes from the nucleotide inposition 10 to the nucleotide in position 49 of said nucleic acid offormula (I).

It has also been shown in the examples that each of the nucleic acids ofsequences SEQ ID NOS. 37 to 39 has the ability to bind to activeGLA-domain coagulation proteins. The aptamer of sequence SEQ ID NO. 39can also be denoted “Mapt-2”. The aptamer of sequence SEQ ID NO. 37 canalso be denoted “Mapt-2-CS”. The aptamer of sequence SEQ ID NO. 38 canalso be denoted “Mapt-2.2.-CS”. It is specified that the sequence of theMapt-2-CS aptamer is included in the sequence of the Mapt-2 aptamer: itis the “core sequence” SEQ ID NO. X of the Mapt-2 aptamer.

It has been shown in the examples that the anti-GLA aptamers of theinvention can be successfully used for producing affinity chromatographysupports, that are of use for separating active GLA-domain coagulationproteins from inactive GLA-domain coagulation proteins.

To prepare affinity chromatography supports in particular, the anti-GLAnucleic aptamers of the invention are preferentially included in achemical structure, also called “compound” in the present description,which comprises in particular a spacer means and, where appropriate, ameans for immobilization on a solid support.

In certain embodiments, the nucleic aptamer is included in the structureof a compound of formula (II) below:[IMM]_(x)-[SPAC]_(y)-[APT]  (II),in which:

-   -   [IMM] signifies a compound for immobilization on a support,    -   [SPAC] signifies a spacer chain,    -   [APT] signifies an anti-GLA aptamer as defined in the present        description,    -   x is an integer equal to 0 or 1, and    -   y is an integer equal to 0 or 1.

In certain embodiments of the compound of formula (II), [APT] consistsof a deoxyribonucleic acid of which the sequence is chosen from thesequences SEQ ID NOS. 3, 4 and 6 to 39.

The “spacer chain” denoted [SPAC] in the compound of formula (II) can beof any known type. The function of said spacer chain is to physicallydistance the nucleic acid from the surface of the solid support on whichsaid compound can be immobilized and to allow a relative mobility of thenucleic acid relative to the surface of the solid support on which itmay be immobilized. The spacer chain limits or prevents sterichindrances, due to too great a proximity between the solid support andthe nucleic acid, from impairing the binding events between said nucleicacid and coagulation protein molecules that may be brought into contacttherewith.

In the compound of formula (II), the spacer chain is preferentiallylinked to the 5′ end or to the 3′ end of the nucleic acid aptamer.

Advantageously, the spacer chain is linked both to one end of theaptamer and to the solid support. This construction with a spacer hasthe advantage of not directly mobilizing the aptamer on the solidsupport. Preferably, the spacer chain is a nonspecific oligonucleotideor polyethylene glycol (PEG) or another hydrophilic hydrocarbon-basedchain. When the spacer chain consists of a nonspecific oligonucleotide,said oligonucleotide advantageously comprises at least 5 nucleotides inlength, preferably between 5 and 15 nucleotides in length.

In the embodiments of a compound of formula (II) in which the spacerchain consists of a polyethylene glycol, said spacer chain encompasses apolyethylene glycol of PEG (C18) type, sold, for example, by the companySigma Aldrich.

As is illustrated in the examples, the purification of active GLA-domaincoagulation proteins is carried out both with compounds of formula (II)comprising a spacer chain [SPAC] and with compounds of formula (II)which do not have a spacer chain [SPAC].

In order to immobilize the aptamer on the spacer chain, the nucleic acidmay be chemically modified with various chemical groups, such as groupswhich make it possible to covalently immobilize said nucleic acid, forinstance thiols, amines or any other group capable of reacting withchemical groups present on the solid support.

In the compound of formula (II), the compound [IMM] consists of acompound chosen from (i) a compound capable of forming one or morecovalent bond(s) with the solid support material and (ii) a compoundcapable of binding specifically on the solid support by means of weaknoncovalent bonds, including hydrogen bonds, electrostatic forces or Vander Waals forces.

In certain cases, the compound [IMM], because it consists of a chain ofatoms linked to one another via covalent bonds, can behave as a spacerchain. However, by definition, a compound [IMM] can never signify a[SPAC] chain in a compound of formula (II) according to the invention.In other words, in a compound of formula (II), the [SPAC] chain, when itis present, cannot be directly linked to the chromatography support,whether via covalent bonds or weak noncovalent bonds.

The first type of compound [IMM] encompasses bifunctional couplingagents, such as glutaraldehyde, SIAB or else SMCC.

The SIAB compound, described by G. T. Hermanson (1996, Bioconjugatetechniques, San Diego: Academic Press, pp 239-242), is the compound offormula (A) below:

The SIAB compound comprises two reactive groups, respectively aniodoacetate group and a sulfo-NHS ester group, these groups reactingrespectively with amino and sulfhydryl groups.

The SMCC compound, which is described by M. K. Samoszuk et al. (1989,Antibody, Immunoconjugates Radiopharm., 2(1):37-46), is the compound offormula (B) below:

The SMCC compound comprises two reactive groups, respectively asulfo-NHS ester group and a maleimide group, which react respectivelywith an amino group and a sulfhydryl group.

The second type of compound [IMM] encompasses biotin, which is capableof binding in a specifically noncovalent manner to avidin orstreptavidin molecules present on the solid support.

Once immobilized on the solid support via the spacer, the anti-GLAaptamer is advantageously modified at its free end (end not linked tothe spacer) by virtue of, and without being limited thereto, achemically modified nucleotide (such as 2′-O-methyl- or2′-fluoro-pyrimidine, 2′-ribopurine, phosphoramidite), an invertednucleotide or a chemical group (PEG, polycations, cholesterol). Thesemodifications make it possible to protect the anti-GLA aptamer againstenzymatic degradations.

The solid support may be an affinity chromatography column composed of agel derived from agarose or cellulose or a synthetic gel such as anacrylamide, methacrylate or polystyrene derivative, a chip such as achip suitable for surface plasmon resonance, a membrane such as apolyamide, polyacrylonitrile or polyester membrane, or a magnetic orparamagnetic bead.

The present invention also relates to a complex between:

-   -   (i) a nucleic aptamer chosen from a nucleic acid of formula (I),        and a compound of formula (II), and    -   (ii) an active GLA-domain coagulation protein, including a        coagulation protein of which the GLA domain is correctly        gamma-carboxylated.

A subject of the present invention is also a support for immobilizing anactive GLA-domain coagulation protein, including a coagulation proteinwith a correctly gamma-carboxylated GLA domain, characterized in that itcomprises a solid support material on which a plurality of moleculeseach consisting of, or each comprising, a nucleic aptamer are grafted,said molecules being chosen from (a) a nucleic acid of formula (I), and(b) a compound of formula (II).

The support above can be used practically in all the applications forwhich it is sought to immobilize an active GLA-domain coagulationprotein, including a human active GLA-domain coagulation protein, whichencompasses applications for the purposes of purifying said activeGLA-domain coagulation protein and applications for the purposes ofdetecting said active GLA-domain coagulation protein.

The preparation of supports on which nucleic aptamers of the inventionwhich bind specifically to an active GLA-domain coagulation protein areimmobilized, is widely illustrated in the examples, in which theaptamers of the invention are used in particular as capture agents, thatcan be used for purifying or for detecting an active GLA-domaincoagulation protein, including a human active GLA-domain coagulationprotein, in samples.

The present invention therefore also relates to a method forimmobilizing an active GLA-domain coagulation protein on a support,comprising a step during which a sample comprising at least oneGLA-domain protein is brought into contact with a solid support on whicha substance chosen from a nucleic acid of formula (I), and a compound offormula (II), has been previously immobilized. Said method may comprise,depending on the technical objectives pursued, an additional step ofrecovering the immobilized active GLA-domain coagulation proteinmolecule(s), complexed with the nucleic acid molecules of formula (I).The additional step of recovering the active GLA-domain coagulationprotein, or the active GLA-domain coagulation proteins, preferentiallyconsists of a step of bringing the complexes of active GLA-domaincoagulation protein(s) with the nucleic acids of formula (I) intocontact with a metal-cation-chelating agent, such as EDTA.

For carrying out affinity chromatography protein purification methodsusing solid supports on which the aptamers of interest are immobilized,those skilled in the art may refer to the work described by Romig et al.(1999, J Chromatogr B Biomed Sci Apl, Vol. 731(2): 275-284).

In addition, the production of an affinity support comprising a nucleicaptamer of the invention and the carrying out of a method for purifyingactive human Factor IX with said affinity support are illustrated in theexamples.

Generally, the solid supports on which the aptamers of the invention canbe immobilized encompass any type of support having the structure andthe composition commonly found for filter supports, of silicon supportfor chips, membranes, etc. The solid supports encompass in particularresins, resins for affinity chromatography columns, polymer beads,magnetic beads, etc. The solid supports also encompass in particularglass-based or metal-based materials, such as steel, gold, silver,aluminum, copper, silicon, glass or ceramic. The solid supports alsoencompass in particular polymer materials, such as a polyethylene, apolypropylene, a polyamide, a polyvinylidene fluoride, and combinationsthereof.

The solid support may be coated with a material that facilitates theattachment, the binding, the formation of complexes, the immobilizationor the interaction with the aptamers.

In certain embodiments, the solid support is a glass slide of which thesurface is coated with a layer of gold, with a layer having undergone atreatment by carboxymethylation, with a layer of dextran, of collagen,of avidin, of streptavidin, etc.

In this way, the aptamers according to the invention can be immobilizedon the solid support by means of an attachment coating, for instancedescribed above, or by chemical reaction with the creation of covalentbonds, or by association via noncovalent bonds, such as hydrogen bonds,electrostatic forces, Van der Waals forces, etc.

The examples describe embodiments of solid supports on which theaptamers of the invention are immobilized via noncovalent bonds.

In the examples, solid supports consisting of a glass slide coated witha layer of streptavidin molecules, and aptamers of the inventionconjugated to a biotin molecule, which are immobilized on the support bynoncovalent biotin/streptavidin association, are in particulardescribed.

In the examples, solid supports consisting of a polystyrene materialcoated with a layer of streptavidin molecules, and aptamers of theinvention conjugated to a biotin molecule, which are immobilized on thesupport by noncovalent biotin/streptavidin association, are alsodescribed.

In certain embodiments, the aptamers of the invention can be immobilizedon a solid support suitable for affinity chromatography,electrochromatography and capillary electrophoresis, as described, forexample, by Ravelet et al. (2006, J Chromatogr A, Vol. 117(1): 1-10),Connor et al. (2006, J Chromatogr A, Vol. 111(2): 115-119), Cho et al.(2004, Electrophoresis, Vol. 25 (21-22): 3730-3739) or else Zhao et al.(2008, Anal Chem, Vol. 80(10): 3915-3920).

An aptamer of formula (I) which is at least 15 nucleotides in length andwhich binds specifically to GLA-domain proteins, or a compound offormula (II), can also be advantageously used as an agent for capturingactive GLA-domain protein(s) in detection or diagnostic methods anddevices.

According to yet another aspect, the present invention also relates to amethod for detecting the presence of one or more active GLA-domaincoagulation protein(s) in a sample, comprising the following steps:

-   -   a) bringing (i) a nucleic acid of formula (I), or a compound of        formula (II) or a solid support on which a plurality of        molecules of said nucleic acid or of said compound are        immobilized, into contact with (ii) said sample; and    -   b) detecting the formation of complexes between (i) said nucleic        acid of formula (I), or said compound of formula (II) or said        support, and (ii) the active GLA-domain coagulation protein(s).

The examples of the present patent application provide variousembodiments of methods for detecting active human FIX/FIXa with aptamersof the invention immobilized beforehand on a solid support.

For carrying out a detection method according to the invention, thesolid support used may be a solid support chosen from the solid supportspreviously described in relation to the method for purifying activeGLA-domain coagulation proteins.

For carrying out a method or preparing a device for detecting activeGLA-domain coagulation proteins, those skilled in the art may refer inparticular to the techniques described in European patent applicationNo. EP 1 972 693, PCT application No. WO 2008/038696, PCT applicationNo. WO 2008/025830, or else PCT application No. WO 2007/0322359.

In certain embodiments, step b) of detecting the formation of complexesbetween (i) said nucleic acid or said solid support and (ii) the activeGLA-domain coagulation protein(s) can be carried out by measuring thesurface plasmon resonance signal, as is described in the examples.

In certain other embodiments, step b) of detecting the formation ofcomplexes between (i) said nucleic acid or said solid support and (ii)the active GLA-domain coagulation protein(s) can be carried out bybringing said complexes optionally formed into contact with a ligandspecific for a GLA-domain coagulation protein, said ligand beingdetectable. The examples describe these embodiments in which use ismade, as detectable ligand of a GLA-domain coagulation protein, ofanti-GLA-domain coagulation protein monoclonal or polyclonal antibodieslabeled with an enzyme, in the case in point horseradish peroxidase, asis conventionally used in tests of ELISA type. As antibodies specificfor GLA-domain coagulation proteins, it is possible to use, depending onthe objectives that are pursued, (i) either antibodies directedspecifically against a predetermined GLA-domain coagulation protein, forexample anti-human FII, anti-human FVII, anti-human FIX, anti-human FX,anti-human protein C or anti-human protein S antibodies, (ii) orantibodies directed against a GLA domain, which are capable ofrecognizing a plurality of GLA-domain proteins, as described, forexample, in U.S. Pat. No. 7,439,025.

Surprisingly, it is shown according to the invention that it is possibleto produce an affinity support as defined in the present description byusing, as anti-GLA nucleic aptamers, DNA aptamers that are neverthelessconsidered in the prior art to be nucleic acid ligands which aredifficult to use and the specificity of which for the target protein isless than the specificity of the RNA molecule of the correspondingsequence. In particular, it is accepted in the prior art that DNAligands have less flexibility than the corresponding RNA and that,consequently, are less capable than RNA ligands of undergoingconformational changes. It is recalled that, when a nucleic aptamerbinds to the target protein, a conformational change takes place. It hasalso been described that the faster the conformational change of thenucleic aptamer, the higher the affinity of said nucleic aptamer for thetarget protein (Michaud et al., 2003, Anal Chem, Vol. 76: 1015-1020);Brumbt et al., 2005, Anal Chem, Vol. 77: 1993-1998).

Thus, in certain embodiments of an affinity support according to theinvention, the anti-GLA aptamers consist of DNA aptamers.

Consequently, in certain embodiments of an affinity support according tothe invention, said immobilized nucleic aptamers, where appropriateincluded in the structure of a compound of formula (I), consist ofdeoxyribonucleic acids.

In certain other embodiments of an affinity support according to theinvention, a first part of said nucleic acids consists ofdeoxyribonucleic acids and the remaining part consists of ribonucleicacids.

Another advantage of the nucleic aptamers concerns the ease with whichthey are produced, compared with the difficulties in synthesizing RNAaptamers, and also their cost price, which is significantly lower thanthe cost price of an RNA aptamer.

These specific embodiments are illustrated in the examples.

A subject of the present invention is also an affinity chromatographydevice for purifying an active GLA-domain coagulation protein, or forpurifying a plurality of active GLA-domain coagulation proteins,comprising a container in which a suitable amount of an affinity supportas defined in the present description is placed.

Varied forms of containers for chromatography supports are known in theprior art and are encompassed by the meaning of the term “container”above. The important characteristics of such a container encompass thepresence of a means of feeding the affinity chromatography device with astarting sample and a means for the liquid to leave after it has beenbrought into contact with the affinity support.

A subject of the present invention is also a method for immobilizing anactive GLA-domain coagulation protein on a support, comprising a stepduring which a sample containing said coagulation protein is broughtinto contact with an affinity support as defined above.

The starting samples from which one or more active GLA-domaincoagulation proteins are purified with an affinity support according tothe invention encompass any type of liquid solution in which said activeGLA-domain coagulation protein(s) is (are) in suspension or is (are)solubilized. Specific embodiments of such samples, in particular inrelation to the purification method described hereinafter, will bedefined subsequently in the present description.

In certain preferred embodiments, said sample contains a human activeGLA-domain coagulation protein. Advantageously, in these embodiments,the sample containing an active GLA-domain coagulation protein ofinterest consists of a liquid sample which contains said protein ofinterest, including a liquid sample comprising said active GLA-domaincoagulation protein and which is capable of also containing (i) inactivemolecules of said GLA-domain coagulation protein and/or (ii) moleculesof the homologous GLA-domain coagulation protein of a nonhuman mammal.In certain embodiments of the purification method above, said sampleconsists of a biological solution, such as a body fluid, a cell, groundcell material, a tissue, ground tissue material, an organ or a wholeorganism.

It has been shown in the examples that an anti-GLA aptamer according tothe invention makes it possible to purify a recombinant human Factor IXproduced in the milk of a pig transgenic for said human Factor IX, owingto the fact that said anti-GLA aptamer binds selectively to the activehuman Factor IX and (i) does not bind to the inactive human Factor IXand (ii) does not bind to the pig Factor IX, whether said pig Factor IXis active or whether said pig Factor IX is inactive.

In certain embodiments of the purification method above, said sampleconsists of a liquid biological solution originating from an animal,such as blood, a blood derivative, mammalian milk or a mammalian milkderivative. Said sample may consist of plasma, plasma cryoprecipitate,clarified milk or derivatives thereof.

In particularly preferred embodiments of the purification method above,said sample originates from an animal transgenic for the humanGLA-domain coagulation protein of interest. Advantageously, the solutionis milk from a mammal or a derivative of milk from a mammal transgenicfor said human protein of interest. For the purpose of the invention,the transgenic animals encompass (i) nonhuman mammals such as cows,goats, rabbits, pigs, monkeys, rats or mice, (ii) birds, or else (iii)insects such as mosquitoes, flies or silkworms. In certain preferredembodiments, the animal transgenic for the human protein of interest isa nonhuman transgenic mammal, entirely preferably a doe rabbittransgenic for said human protein of interest. Advantageously, thetransgenic mammal produces said recombinant human protein of interest inits mammary glands, owing to the insertion into its genome of anexpression cassette comprising a nucleic acid encoding said protein ofinterest which is placed under the control of a specific promoterallowing the expression of the transgenic protein in the milk of saidtransgenic mammal.

A method for producing said human GLA-domain protein in the milk of atransgenic animal can comprise the following steps: a DNA moleculecomprising a gene encoding the protein of interest, said gene beingunder the control of a promoter of a protein naturally secreted in milk(such as the casein promoter, the beta-casein promoter, the lactalbuminpromoter, the beta-lactoglobulin promoter or the WAP promoter), isintegrated into an embryo of a nonhuman mammal. The embryo is thenplaced in a female mammal of the same species. Once the mammal resultingfrom the embryo has developed sufficiently, lactation is induced in themammal, and then the milk is collected. The milk then contains therecombinant human protein of interest.

An example of a method for preparing protein in the milk of a female ofa mammal other than a human being is given in document EP 0 527 063, theteaching of which can be reproduced for producing the protein of theinvention. A plasmid containing the WAP (Whey Acidic Protein) promoteris produced by introducing a sequence comprising the promoter of the WAPgene, this plasmid being produced in such a way as to be able to receivea foreign gene placed under the control of the WAP promoter. The plasmidcontaining the promoter and the gene encoding the protein of theinvention are used to obtain transgenic doe rabbits, by microinjectioninto the male pronucleus of doe rabbit embryos. The embryos are thentransferred into the oviduct of hormonally prepared females. Thepresence of the transgenes is revealed by the Southern technique, usingthe DNA extracted from the young transgenic rabbits obtained. Theconcentrations in the milk of the animals are evaluated by means ofspecific radioimmunological tests.

Other documents describe methods for preparing proteins in the milk of afemale of a mammal other than a human being. Mention may be made,without being limited thereto, of the documents U.S. Pat. No. 7,045,676(transgenic mouse) and EP 1 739 170 (production of von Willebrand factorin a transgenic mammal).

The purification method of the invention is also perfectly suitable forobtaining an active GLA-domain coagulation protein purified from asample of human blood plasma, or from a fraction of human blood plasma,for example the cryoprecipitated fraction of human blood plasma.

In certain embodiments of the purification method above, the targetactive GLA-domain coagulation protein is human.

In certain embodiments of the purification method above, the samplecomprises at least one nonhuman active GLA-domain coagulation protein.

In certain embodiments of the purification method above, said humanactive GLA-domain coagulation protein is homologous to said nonhumanGLA-domain protein.

In certain embodiments of the purification method above, said humanactive GLA-domain coagulation protein is the homologue of said nonhumanGLA-domain protein.

In certain embodiments of the purification method above, the startingsample may consist of the crude material, which is either the sample ofhuman blood plasma, or a fraction thereof, or the body fluid of anonhuman mammal transgenic for the GLA-domain protein of interest, andwhich contains said GLA-domain protein of interest to be purified. Thebody fluids of a transgenic nonhuman mammal encompass the milk or afraction of the milk, for example a defatted fraction of the milk orelse a fraction depleted of casein micelles.

However, the embodiment above is not the preferred embodiment of thepurification method of the invention, in particular owing to the risk ofclogging of the affinity support by the numerous proteins present in thecrude starting sample.

In preferred embodiments, said starting sample consists of a liquidsolution containing the active GLA-domain coagulation protein ofinterest in suspension in said solution, said liquid solution consistingof an intermediate product generated during a method for multisteppurification of an active GLA-domain coagulation protein.

By way of illustration, for a method for purifying an active GLA-domaincoagulation protein from a body fluid of a nonhuman mammal transgenicfor said protein, the starting sample may consist of an eluate from ahydrophobic interaction chromatography step, such as a chromatographystep in which a chromatographic support of the MEP HyperCel® type isused. This particular embodiment of a purification method according tothe invention is illustrated in the examples.

In the same way, for a method for purifying the active GLA-domaincoagulation protein of interest from human plasma, the starting samplemay consist of a filtrate from a deep-filtration step carried out on thecryoprecipitated fraction of a human plasma sample.

Generally, the conditions for using the affinity support in order tocarry out the purification method of the invention are very similar tothe conventional conditions for using a conventional chromatographysupport, for example of the immunoaffinity support type on which ligandantibodies are immobilized. Those skilled in the art may, for example,refer to the handbook by Bailon et al. (Pascal Bailon, George K.Ehrlich, Wen-Jian Fung and Wolfgang Berthold, An Overview of AffinityChromatography, Humana Press, 2000).

However, as will be detailed in the description that follows, theconditions of elution step c) of the method of the invention are veryadvantageous for the purification of an active GLA-domain coagulationprotein.

In step a), an appropriate volume of the sample to be purified isbrought into contact with the affinity support. Complexes are formedbetween (i) the anti-GLA aptamers immobilized on said affinity supportand (ii) the active GLA-domain coagulation protein of interest containedin the sample to be purified.

It has been shown in the examples that the conditions for binding of theanti-GLA aptamers to the active GLA-domain coagulation proteins arepromoted in the presence of calcium, for example in the form of calciumchloride.

Thus, in certain embodiments of step a), a buffer solution comprising afinal concentration of CaCl₂ of at least 1 mM is used, which encompassesat least 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM,12 mM, 13 mM, 14 mM and 15 mM. In step a), a buffer solution comprisinga final concentration of CaCl₂ of at most 50 mM is advantageously used.

It has also been shown in the examples that the conditions for bindingof the anti-GLA aptamers to the active GLA-domain coagulation proteinsare promoted in the absence of magnesium chloride.

Thus, in certain embodiments of step a), a buffer solution which doesnot comprise magnesium chloride is used.

By way of illustration, it has been shown in the examples that, for achromatography support on which an anti-GLA aptamer according to theinvention is immobilized, the affinity of said support for a GLA-domaincoagulation protein is increased practically by a factor of ten when abuffer solution devoid of magnesium chloride is used in step a), in thecase in point a buffer solution of 50 mM Tris-HCl and CaCl₂ at pH 7.5,in comparison with a buffer solution that is identical but comprisesmagnesium chloride.

In preferred embodiments of the method, at the end of step a) and priorto step b) described in detail later, one or more steps of washing theaffinity support are carried out so as to remove the substances whichare not specifically retained, for example the substances simplyadsorbed onto said support. A conventional washing buffer well known tothose skilled in the art can be used.

However, in certain embodiments of the washing step(s), a washing buffercomprising NaCl, and/or ethylene glycol, and/or propylene glycol, and/orethanol, can be used.

It has been shown in the examples that the use of a washing solutioncomprising NaCl does not lead to any modification in the specificbinding of the GLA-domain coagulation proteins to the immobilizedanti-GLA aptamers. It has been shown in particular that a final NaClconcentration of 3M does not disrupt said specific binding.

Thus, in certain embodiments of the washing step(s) prior to step b), awashing solution having a final NaCl concentration of at least 0.5M,which includes at least 1M, 1.5M, 2M, 2.5M and 3M, is used. The finalNaCl concentration is advantageously at most 4M.

The results of the examples thus show that the ability of the anti-GLAaptamers of the invention to bind to the GLA-domain coagulation proteinsis not modified when the complexes formed between the immobilizedaptamers and the GLA-domain protein(s) are subjected to an environmentof high ionic strength, this being an entirely unexpected result. It isin fact recalled that it is common to resort to a buffer of high ionicstrength as a buffer for eluting substances complexed beforehand toimmobilized ligands of an affinity support, including an affinitysupport comprising immobilized aptamers.

The results of the examples thus show that the anti-GLA aptamersaccording to the invention have specific and unexpected characteristicswith regard to the absence of effect of a high ionic strength on theirability to bind to an active GLA-domain coagulation protein.

Without wishing to be bound by any theory, the applicant thinks thatthese unexpected properties of the anti-GLA aptamers of the inventionare a consequence of the ability of said anti-GLA aptamers to bindspecifically to the GLA domain which is common to active GLA-domaincoagulation proteins. In particular, the applicant thinks that theanti-GLA aptamers of the invention bind to the target proteins by meansof divalent noncovalent bridges which can be generated only at the levelof the GLA domain, and that the creation of said divalent bridgesprevents the subsequent binding of the ions originating from the NaCl onthe GLA domain.

It has also been shown in the examples that the use of a washingsolution comprising ethylene glycol does not lead to any modification inthe specific binding of the active GLA-domain coagulation proteins tothe immobilized anti-GLA aptamers. It has been shown in particular thata final ethylene glycol concentration of 1.5M does not disrupt saidspecific binding.

Thus, in certain embodiments of the washing step(s) prior to step b), awashing solution having a final ethylene glycol concentration of atleast 0.5M, which includes at least 1M and 1.5M, is used.

It has also been shown in the examples that the use of a washingsolution comprising propylene glycol does not lead to any modificationin the specific binding of the GLA-domain coagulation proteins to theimmobilized anti-GLA aptamers. It has been shown in particular that afinal propylene glycol concentration of 50% (v/v) does not disrupt saidspecific binding.

Thus, in certain embodiments of the washing step(s) prior to step b), awashing solution having a final propylene glycol concentration of atleast 10% (v/v), which includes at least 15%, 20%, 25%, 30%, 35%, 40%,45% and 50%, is used.

It has also been shown in the examples that the use of a washingsolution comprising ethanol does not lead to any modification in thespecific binding of the active GLA-domain coagulation proteins to theimmobilized anti-GLA aptamers. It has been shown in particular that afinal ethanol concentration of 10% (v/v) does not disrupt said specificbinding.

Thus, in certain embodiments of the washing step(s) prior to step b), awashing solution having a final ethanol concentration of at least 1%(v/v), which includes at least 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.50,5.00, 5.50, 6.00, 6.50, 7.00, 7.50, 8.00, 9.00, 9.5% and 10.0%, is used.

It has been shown that such a washing buffer leads to drastic conditionsfor removing the substances that are not specifically retained on theaptamers, while at the same time preserving the specific binding of themolecules of the active GLA-domain coagulation protein(s) to theaptamers immobilized on the affinity support. It is recalled that such atechnical advantage linked to the exclusive or virtually exclusiveremoval of the substances not specifically bound to the ligandsimmobilized on the affinity support cannot be easily realized with anaffinity support on which antibodies are immobilized.

Step b) consists of a step of eluting the molecules of the activeGLA-domain coagulation protein of interest having formed complexes withthe anti-GLA nucleic aptamers during step a).

As is illustrated in the examples, a specific advantage of thepurification method above is the possibility of carrying out the elutionstep by bringing the complexes formed between (i) the anti-GLA nucleicaptamers immobilized on said affinity support and (ii) said activeGLA-domain coagulation protein into contact with adivalent-ion-chelating agent, such as EDTA.

This technical advantage, which is made possible by virtue of thecharacteristics of the affinity support of the invention, makes itpossible to elute the active GLA-domain coagulation protein withoutrequiring any recourse to the use of drastic elution conditions, thatmay denature, at least partially, the active GLA-domain coagulationprotein of interest. Said drastic conditions which are avoided encompassthe use of an acidic pH for the elution step, which is commonlyperformed for the methods for purifying proteins on affinity supportsthat are known, and most particularly on affinity supports comprisingimmobilized antibodies.

Thus, in certain embodiments of the purification method above, step b)is carried out by bringing the affinity support into contact with anelution buffer containing a divalent-ion-chelating agent, preferablyEDTA.

By way of illustration, the elution buffer may contain a final EDTAconcentration of at least 1 mM and of at most 100 mM.

The expression “at least 1 mM” encompasses at least 2, 3, 4, 5, 6, 7, 8,9 or 10 mM.

The expression “at most 100 mM” encompasses at most 29, 28, 27, 26, 25,24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12 or 11 mM.

In step c), the active GLA-domain protein of interest is purified bycollecting the eluate liquid obtained at the end of step b).

At the end of step c), a purified liquid composition of the coagulationprotein of interest is obtained. Said purified liquid composition canthen be treated appropriately, according to any known technique forconditioning and storing proteins, including by direct bottling orbottling after dilution with a suitable solution, or else byfreeze-drying, preferentially under sterile and apyrogenic conditions,and then storage under appropriate conditions, at ambient temperature,at −4° C. or else at a low temperature, depending on the type ofconditioning selected.

As has already been mentioned previously in the present description, theaffinity support of the invention can, with the successive cycles of usefor purifying an active GLA-domain coagulation protein of interest,experience a reduction in its absorption capacity, for example owing tothe fact that elution step c) does not make it possible tosystematically release all of the molecules of coagulation protein,thereby reducing the number of free aptamer sites for the subsequentpurification cycles.

As for all known chromatography supports, it is therefore necessary, atappropriate moments, to carry out a step of regenerating the affinitysupport, in order to release all of the molecules of active GLA-domaincoagulation protein from said support, and to remove any substance thatmay be bound to the solid material of the affinity support, generally bynonspecific binding.

Thus, in certain embodiments, the purification method of the inventioncomprises an additional step d) of regenerating the affinity support bybringing said affinity support into contact with a regeneratingsolution.

Varied buffers for regenerating chromatography supports, in particularaffinity chromatography supports, are well known to those skilled in theart, and can be used in step d) of the method. Those skilled in the artmay refer, for example, to the handbook by Mohr et al. (AffinityChromatography: Practical and Theoretical Aspects, Peter Mohr, KlausPommerening, Edition: illustrated, CRC Press, 1985).

By way of illustration, step d) of regenerating the affinity support canbe carried out by bringing said support into contact with a buffersolution of 20 mM Tris, 5% polyethylene glycol and 1M NaCl, for exampleat pH 7.5, as is illustrated in the examples.

The purification method above makes it possible to obtain an activeGLA-domain coagulation protein at a very high degree of purity,optionally at a degree of purity greater than 99.95% by weight, relativeto the total weight of the proteins contained in the purified finalproduct.

Another advantage of the purification method above, in particular in theembodiments in which the starting sample consists of a sample comprisingthe human active GLA-domain coagulation protein of interest inrecombinant form as a mixture with proteins naturally produced by thenonhuman transgenic mammal, is that the final composition comprising therecombinant human protein of interest at a high degree of purity issubstantially free of proteins originating from said transgenic mammal,and in particular substantially free of proteins of said mammal whichare homologues of said recombinant human active GLA-domain coagulationprotein.

The present invention also relates to a nucleic aptamer which bindsspecifically to biologically active GLA-domain proteins, of which theability to bind to a target GLA-domain protein is not modified by anenvironment of high ionic strength.

The invention relates in particular to a nucleic aptamer which bindsspecifically to biologically active GLA-domain proteins, of which theability to bind to a target biologically active GLA-domain protein isnot modified by an environment of high ionic strength having a finalNaCl concentration of at least 0.5M.

The biologically active GLA-domain proteins encompass biologicallyactive GLA-domain coagulation proteins.

Said “environment” at high ionic strength encompasses a buffer solutionat high ionic strength.

An environment of high ionic strength includes an environment having afinal NaCl concentration of at least 1M, 1.5M, 2M, 2.5M and 3M. Thefinal NaCl concentration is advantageously at most 4M.

The expression “is not modified” means that the binding between thetarget GLA-domain protein and the nucleic aptamer which bindsspecifically to biologically active GLA-domain proteins withstands anenvironment of high ionic strength. For example, at least 80% of thetarget biologically active GLA-domain proteins remain bound to thenucleic aptamer which binds specifically to biologically activeGLA-domain proteins in an environment at high ionic strength, preferably85%, 90%, 95%, 96%, 97%, 98%. In particular, this means that a washingstep in an environment of high ionic strength does not result in theelution of more than 20% of the biologically active GLA-domain proteinsbound to the nucleic aptamer which binds specifically to biologicallyactive GLA-domain proteins, preferably of more than 10%, 5%, 4%, 3%, 2%,1%.

In one particular embodiment, the nucleic aptamer according to theinvention also has the ability to bind to a target biologically activeGLA-domain protein without being modified by a hydrophobic environment,such as a 10% ethanol or 50% propylene glycol solution.

These properties can advantageously be used for very efficiently washingan affinity support on which a nucleic aptamer according to theinvention is immobilized, thereby making it possible to obtain betterremoval of the contaminants nonspecifically bound to the column. Theimprovement in the washing efficiency makes it possible to increase thepurity of the biologically active GLA-domain proteins that it is soughtto purify.

As has been described previously in the present description and is alsoillustrated in the examples, the complexes formed between the nucleicaptamers of the invention and the target biologically active GLA-domainproteins can be dissociated by bringing said complexes into contact witha solution comprising a divalent-ion-chelating agent, for example EDTA.Thus, according to another of their characteristics, the nucleicaptamers of the invention consist of nucleic aptamers which allow theformation of complexes with target GLA-domain proteins, it beingpossible for said complexes to be dissociated, with release of thetarget biologically active GLA-domain proteins, by bringing saidcomplexes into contact with a medium comprising a divalent-ion-chelatingagent, for example EDTA.

As already specified previously, a complex between a nucleic aptamer ofthe invention and a target biologically active GLA-domain protein can bedissociated by bringing said complex into contact with a medium,including a buffer solution, comprising a final EDTA concentration of atleast 1 mM and of at most 100 mM.

As is illustrated in the examples, advantage is taken of the ability ofcertain embodiments of the anti-GLA aptamers of the invention to bind toa diversity of active GLA-domain coagulation proteins, for example to adiversity of human active GLA-domain coagulation proteins, forsimultaneously purifying a plurality of active GLA-domain coagulationproteins that may be contained in the starting sample, using a singleaffinity chromatography support. For example, use may be made of theprocess for purifying a GLA-domain protein above for simultaneouslypurifying several GLA-domain coagulation factors contained in thestarting sample, for example for simultaneously purifying the FactorsVII, IX and X contained in a cryoprecipitated fraction of human bloodplasma.

A subject of the present invention is also a purified composition of arecombinant human active GLA-domain coagulation protein comprising atleast 99.9% by weight of said recombinant human active GLA-domainprotein and which is substantially free of nonhuman proteins.

The present invention also relates to a purified composition of arecombinant human active GLA-domain coagulation protein comprising atleast 99.9% by weight of said recombinant human GLA-domain protein andat most 0.1% by weight of nonhuman proteins, the percentages by weightbeing expressed relative to the total weight of proteins of saidpurified composition.

In the purified composition above, “at least 99.9%” encompasses at least99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98% and99.99%.

In the purified composition above, “at most 0.1%” encompasses at most0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02% and 0.01%.

The present invention also relates to a purified composition as definedabove, that can be used as a medicament.

The invention also relates to a pharmaceutical composition comprising apurified composition of a recombinant human active GLA-domaincoagulation protein as defined above, in combination with one or morepharmaceutically acceptable excipients.

The invention also relates to a purified composition as defined above,for treating coagulation disorders.

The invention also relates to the use of a purified composition asdefined above, for producing a medicament for treating coagulationdisorders.

Methods for Obtaining Anti-GLA Aptamers

Generally, an anti-GLA aptamer according to the invention can beobtained according to a method based on the general principles of thetechnique known as SELEX (Systematic Evolution of Ligands by ExponentialEnrichment), which was initially described in particular in PCTapplication No. WO 1991/019813. The SELEX method for selecting aptamersconsists in bringing a protein into contact with a combinatorial libraryof nucleic acids, DNA or RNA (in general 10¹⁵ molecules); the nucleicacids which do not bind to the target are removed, the nucleic acidswhich bind to the target are isolated and amplified by PCR. The methodis repeated until the solution is sufficiently enriched with the nucleicacids having good affinity for the protein of interest (Tuerk and Gold,“Systematic evolution of ligands by exponential enrichment: RNA ligandsto bacteriophage T4 DNA polymerase” (1990) Science, 249(4968): 505-10and Ellington and Szostak, “In vitro selection of RNA molecules thatbind specific ligands”, (1990) Nature August 30; 346(6287): 818-22).Other SELEX method examples are given in documents EP 0 786 469, EP 668931, EP 1 695 978 and EP 1 493 825, the teachings of which can bereproduced in carrying out the method for selecting a nucleic aptamerused according to the invention. Certain variants of the SELEX methodcomprise steps of counterselection of aptamers previously selected bybinding to the target protein of interest. The counterselection step(s)can consist of a step in which a collection of aptamers, which haspreviously been selected with the target protein of interest, is broughtinto contact with non-target molecules, so as to remove, from thestarting collection of aptamers, those which bind to the non-targetmolecules. The implementation of such a counterselection step, in amethod for obtaining nucleic aptamers, is capable of increasing thespecificity or the affinity of the aptamers selected at the end of themethod.

A first method for obtaining an anti-GLA aptamer can consist of a methodcomprising the following steps:

-   -   a) providing a mixture of nucleic acids, also called        “collection” of nucleic acids having distinct sequences,    -   b) bringing the mixture of nucleic acids provided in step a), or        the mixture of nucleic acids obtained at the end of step d) when        step b) is repeated, into contact with a first target active        GLA-domain coagulation protein, under conditions which allow the        formation of complexes between nucleic acids of said mixture and        said first target GLA-domain coagulation protein,    -   c) carrying out a separation between (i) the nucleic acid(s)        having formed complexes with said first target active GLA-domain        coagulation protein and (ii) the nucleic acids not having formed        complexes with said first target active GLA-domain coagulation        protein,    -   d) amplifying the nucleic acids having formed complexes with        said first target active GLA-domain coagulation protein, so as        to obtain a mixture or a collection of nucleic acids which bind        to said first target active GLA-domain coagulation protein,    -   e) repeating steps b) to d) a sufficient number of times to        obtain a collection of nucleic acids having the desired ability        to bind to said first target active GLA-domain coagulation        protein,    -   f) bringing the mixture of nucleic acids provided in step e), or        the mixture of nucleic acids obtained at the end of step h) when        step f) is repeated, into contact with a second target active        GLA-domain coagulation protein, under conditions which allow the        formation of complexes between nucleic acids of said mixture and        said second target active GLA-domain coagulation protein,    -   g) carrying out a separation between (i) the nucleic acid(s)        having formed complexes with said second target active        GLA-domain coagulation protein and (ii) the nucleic acids not        having formed complexes with said second target active        GLA-domain coagulation protein,    -   h) amplifying the nucleic acids having formed complexes with        said second target active GLA-domain coagulation protein, so as        to obtain a mixture or a collection of nucleic acids which bind        to said second target active GLA-domain coagulation protein,    -   i) repeating steps f) to h) a sufficient number of times to        obtain a collection of nucleic acids having the desired ability        to bind to said second target active GLA-domain coagulation        protein, said nucleic acids having the ability to bind both to        said first target active GLA-domain coagulation protein and to        the second target active GLA-domain coagulation protein,    -   j) optionally, repeating steps f) to i) with one or more other        distinct target active GLA-domain coagulation proteins, so as to        obtain, at the end of the method, a mixture or a collection of        nucleic acids termed “anti-GLA aptamers” having the ability to        bind to the active GLA-domain coagulation proteins.

The nucleic aptamers which are obtained at the end of step j) of themethod above are capable of binding selectively to active GLA-domaincoagulation proteins and do not bind to the other proteins, and inparticular do not bind to the inactive GLA-domain coagulation proteins.

Generally, the detailed protocols for implementing the steps of themethod above can be found by those skilled in the art in the numerouspublications relating to the SELEX technique, including in thereferences previously cited in the present description.

In certain embodiments of the method for obtaining anti-GLA aptamersabove, from 2 to 6 target active GLA-domain coagulation proteins aresuccessively used for selecting anti-GLA aptamers, i.e. nucleic aptamersspecific for active GLA-domain coagulation proteins, but not specificfor a given active GLA-domain coagulation protein. In practice, it hasbeen shown that the successive use, in the method above, of 2 distincttarget active GLA-domain coagulation proteins makes it possible toobtain anti-GLA aptamers.

For implementing the method for obtaining anti-GLA aptamers above, thetarget active GLA-domain coagulation proteins can be chosen from thegroup consisting of Factor II, Factor VII, Factor IX, Factor X, proteinC and protein S. The order in which the target proteins is used is notan essential characteristic of the method above.

According to an alternative, the anti-GLA nucleic aptamers can beobtained according to a second method comprising the following steps:

-   -   a) providing a mixture of nucleic acids, also called        “collection” of nucleic acids having distinct sequences,    -   b) bringing the mixture of nucleic acids provided in step a), or        the mixture of nucleic acids obtained at the end of step d) when        step b) is repeated, into contact with the GLA domain of a first        target active GLA-domain coagulation protein, under conditions        which allow the formation of complexes between nucleic acids of        said mixture and the GLA domain of said first target active        GLA-domain coagulation protein,    -   c) carrying out a separation between (i) the nucleic acid(s)        having formed complexes with the GLA domain of said first target        active GLA-domain coagulation protein and (ii) the nucleic acids        not having formed complexes with the GLA domain of said first        target active GLA-domain coagulation protein,    -   d) amplifying the nucleic acids having formed complexes with the        GLA domain of said first target active GLA-domain coagulation        protein, so as to obtain a mixture or a collection of nucleic        acids which bind to the GLA domain of said first target active        GLA-domain coagulation protein,    -   e) repeating steps b) to d) a sufficient number of times to        obtain a collection of nucleic acids having the desired ability        to bind to the GLA domain of said first target active GLA-domain        coagulation protein,    -   f) bringing the mixture of nucleic acids provided in step e), or        the mixture of nucleic acids obtained at the end of step h) when        step f) is repeated, into contact with the GLA domain of a        second target active GLA-domain coagulation protein, under        conditions which allow the formation of complexes between        nucleic acids of said mixture and the GLA domain of said second        target active GLA-domain coagulation protein,    -   g) carrying out a separation between (i) the nucleic acid(s)        having formed complexes with the GLA domain of said second        target active GLA-domain coagulation protein and (ii) the        nucleic acids not having formed complexes with the GLA domain of        said second target active GLA-domain coagulation protein,    -   h) amplifying the nucleic acids having formed complexes with the        GLA domain of said second target active GLA-domain coagulation        protein, so as to obtain a mixture or a collection of nucleic        acids which bind to the GLA domain of said second target active        GLA-domain coagulation protein,    -   i) repeating steps f) to h) a sufficient number of times to        obtain a collection of nucleic acids having the desired ability        to bind to the GLA domain of said second target active        GLA-domain coagulation protein, said nucleic acids having the        ability to bind both to the GLA domain of said first target        active GLA-domain coagulation protein and to the GLA domain of        the second target active GLA-domain coagulation protein,    -   j) optionally, repeating steps f) to i) with the GLA domain of        one or more other distinct target GLA-domain proteins, so as to        obtain, at the end of the method, a mixture or a collection of        nucleic acids termed “anti-GLA aptamers” having the ability to        bind to the active GLA-domain coagulation proteins.

The nucleic aptamers which are obtained at the end of step j) of themethod above are capable of binding selectively to active GLA-domaincoagulation proteins and do not bind to the other proteins, and inparticular do not bind to the inactive GLA-domain coagulation proteins.

In certain embodiments of the second method for obtaining anti-GLAaptamers above, the GLA domains originating from 2 to 6 target activeGLA-domain coagulation proteins are successively used for selectinganti-GLA aptamers.

For implementing the second method for obtaining anti-GLA aptamersabove, the target active GLA-domain coagulation proteins from which theGLA domains used originate can be chosen from the group consisting ofFactor II, Factor VII, Factor IX, Factor X, protein C and protein S. Theorder in which the target GLA domains are used is not an essentialcharacteristic of the method above.

The essential difference between the second method for obtaininganti-GLA aptamers above and the first method for obtaining anti-GLAaptamers previously described lies in the target, which consists of asuccession of target active GLA-domain coagulation proteins in the firstmethod and consists of a succession of GLA domains of active GLA-domaincoagulation proteins in the second method.

According to the invention, anti-GLA aptamers, which bind to a pluralityof active GLA-domain coagulation proteins, can be obtained according toa third method, the principles of which are identical to the first andsecond methods described above, but in which the target substances arealternately active GLA-domain coagulation proteins and GLA domains ofactive GLA-domain coagulation proteins. The order in which the activeGLA-domain coagulation proteins and the GLA domains of active GLA-domaincoagulation proteins are used is easily determined by those skilled inthe art, according in particular to the target substances to which saidperson skilled in the art has access.

The target GLA domains which are used in each of the second and thirdmethods above can be easily obtained by those skilled in the art, inparticular in situations in which the amino acid sequence of the parentGLA-domain coagulation protein, and therefore also the amino acidsequence of the GLA domain under consideration, are known.

According to certain embodiments, a GLA domain is obtained from theparent GLA-domain coagulation protein by proteolysis of said GLA-domainprotein, using one or more suitable proteolytic enzymes of knowncleavage specificity.

According to other embodiments, a GLA domain is obtained by peptidesynthesis techniques known per se, on the basis of their known aminoacid sequence, and then by gamma-carboxylation of the glutamine residuesof the GLA domain, using a carboxylase, according to techniques wellknown to those skilled in the art. By way of illustration, a peptidecomprising the GLA domain of a GLA-domain coagulation protein can besynthesized with a peptide synthesizer apparatus of the Milligen® 9050Plus type (Perkin Elmer, Stockholm, Sweden), for example usng DPfp Fmocamino acids sold by the company PerSeptive Biosystems® (Framingham,Mass., United States). For the step of enzymatic gamma-carboxylation ofthe neosynthesized GLA domain, use may, for example, be made of asemi-purified carboxylase as described by Soute et al. (1987, ThrombHaemostasis, Vol. 57: 77-81), according to the technique described by Wuet al. (1990, J Biol Chem, Vol. 265(22): 13124-13129).

Other anti-GLA aptamers of the invention, which bind selectively to asingle active GLA-domain coagulation protein, can also be obtainedaccording to other methods which are described below, which also consistof processes of which the principle is based on the implementation ofprotocols used for the SELEX technique.

According to the invention, a fourth method for obtaining anti-GLAaptamers which bind selectively to a single active GLA-domaincoagulation protein has been developed, which method comprises (i) oneor more steps of selecting nucleic acids which bind to an activeGLA-domain coagulation protein, and (ii) one or more steps ofcounterselection in order to remove (ii-a) the nucleic acids which bindto the inactive form of said GLA-domain coagulation protein, and/or(ii-b) the nucleic acids which bind to other GLA-domain coagulationproteins. It is specified that the fourth method below comprises one ormore steps of counterselection of nucleic acids previously selected forbinding to the active GLA-domain coagulation protein of interest.However, the counterselection step(s), or else one or more additionalcounterselection steps, can be provided for prior to step a) of themethod below.

Thus, a fourth method for obtaining anti-GLA aptamers consists of amethod comprising the following steps:

-   -   a) providing a mixture of nucleic acids, also called        “collection” of nucleic acids having distinct sequences, said        collection of nucleic acids optionally having been obtained at        the end of one or more counterselection steps as described        above,    -   b) bringing the mixture of nucleic acids provided in step a), or        the mixture of nucleic acids obtained at the end of step d) when        step b) is repeated, into contact with a first target active        GLA-domain coagulation protein, under conditions which allow the        formation of complexes between nucleic acids of said mixture and        the GLA domain of said first target active GLA-domain        coagulation protein,    -   c) carrying out a separation between (i) the nucleic acid(s)        having formed complexes with said first target active GLA-domain        coagulation protein and (ii) the nucleic acids not having formed        complexes with said first target active GLA-domain coagulation        protein,    -   d) amplifying the nucleic acids having formed complexes with        said first target active GLA-domain coagulation protein, so as        to obtain a mixture or a collection of nucleic acids which bind        to said first target active GLA-domain coagulation protein,    -   e) repeating steps b) to d) a sufficient number of times to        obtain a collection of nucleic acids having the desired ability        to bind to said first target active GLA-domain coagulation        protein,    -   f) bringing the mixture of nucleic acids obtained at the end of        step d), or at the end of step e) when steps b) to d) are        repeated, into contact with an inactive form of said first        target GLA-domain coagulation protein, under conditions which        allow the formation of complexes between nucleic acids of said        mixture and said first target inactive GLA-domain coagulation        protein,    -   g) carrying out a separation between (i) the nucleic acid(s)        having formed complexes with said inactive form of said target        GLA-domain coagulation protein and (ii) the nucleic acids not        having formed complexes with said inactive form of said target        GLA-domain coagulation protein,    -   h) amplifying the nucleic acids not having formed complexes with        said inactive form of said target GLA-domain coagulation        protein, so as to obtain a mixture or a collection of nucleic        acids which bind to said active form of said first GLA-domain        coagulation protein and which do not bind to said inactive form        of said GLA-domain coagulation protein,    -   i) repeating steps f) to h) a sufficient number of times to        obtain a collection of nucleic acids having the desired ability        to bind to said target active GLA-domain coagulation proteins.

In the fourth method for obtaining anti-GLA aptamers above, steps b) toe) consist of steps for selecting nucleic acids which bind to the activeGLA-domain coagulation protein of interest. In said fourth method, stepsf) to i) consist of counterselection steps, during which those of thenucleic acids which also bind to the inactive forms of the activeGLA-domain coagulation protein of interest are removed from thecollection of nucleic acids selected in steps b) to e) for their abilityto bind to the active GLA-domain coagulation protein of interest.

For implementing a SELEX-type method comprising one or more steps ofcounterselection against a non-target protein, those skilled in the artmay refer in particular to the general teaching and also to thetechnical protocols described in U.S. Pat. No. 5,580,737.

In certain embodiments of the fourth method for obtaining anti-GLAaptamers above, counterselection steps f) to i) can be carried out withvarious inactive forms of a single GLA-domain coagulation protein ofinterest, for instance a protein comprising a GLA domain in whichsuccessively one GLA residue and then more than one GLA residue is notgamma-carboxylated and is therefore present in the GLA domain as aglutamine residue. By way of illustration, in order to implementsuccessive counterselection steps, it is possible to successively useforms of the protein of interest comprising a GLA domain in which,successively, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 GLA residues arepresent as glutamine residues, the number of GLAs present as GLU beingof course limited by the maximum number of GLA residues that can bepresent in the GLA domain under consideration when it is completelygamma-carboxylated.

In order to implement the fourth method for obtaining anti-GLA aptamersabove, the GLA-domain coagulation proteins can be chosen from the groupconsisting of Factor II, Factor VII, Factor IX, Factor X, protein C andprotein S.

Other alternatives of a method for selecting aptamers which bindspecifically to active GLA-domain coagulation proteins can be easilydeveloped by those skilled in the art, including selection methods thatinclude one or more initial counterselection step(s) using inactiveGLA-domain coagulation proteins, followed by one or more selectionstep(s) in which active GLA-domain coagulation proteins are used.

According to yet other embodiments, a method for obtaining an anti-GLAaptamer may be of the type of any one of the methods described above, towhich may be added a step of selecting those of the aptamers of whichthe ability to bind to the target protein is not modified by anenvironment of high ionic strength, for example a buffer solutioncomprising NaCl at a final concentration of at least 0.5M, whichincludes at least 1M, 1.5M, 2M, 2.5M and 3M.

According to yet other embodiments, a method for obtaining an anti-GLAaptamer may be of the type of any one of the methods described above, towhich may be added a step of selecting those of the aptamers which allowthe formation of complexes with target GLA-domain proteins, it beingpossible for said complexes to be dissociated, with release of thetarget GLA-domain proteins, by bringing said complexes into contact witha medium comprising a divalent-ion-chelating agent, for example EDTA.The step of selecting these aptamers can be carried out with a medium,including a buffer solution, comprising a final EDTA concentration of atleast 1 mM and at most 100 mM.

According to yet other embodiments, a method for obtaining an anti-GLAaptamer may be of the type of any one of the methods described above, towhich may be added a step of selecting those of the aptamers of whichthe ability to bind the target protein is not modified by the presenceof an alkylene glycol or of a polyalkylene glycol.

According to yet other embodiments, a method for obtaining an anti-GLAaptamer may be of the type of any one of the methods described above, towhich may be added a step of selecting those of the aptamers of whichthe ability to bind to the target protein is not modified by thepresence of ethylene glycol. The step of selecting these aptamers may becarried out with a medium, including a buffer solution, comprising afinal ethylene glycol concentration of at least 0.5M, which includes atleast 1M and 1.5M.

According to yet other embodiments, a method for obtaining an anti-GLAaptamer may be of the type of any one of the methods described above, towhich may be added a step of selecting those of the aptamers of whichthe ability to bind to the target protein is not modified by thepresence of propylene glycol. The step of selecting these aptamers maybe carried out with a medium, including a buffer solution, comprising afinal propylene glycol concentration of at least 10% (v/v), whichincludes at least 15%, 20%, 25%, 30%, 35%, 40%, 45% and 50%.

According to yet other embodiments, a method for obtaining an anti-GLAaptamer may be of the type of any one of the methods described above, towhich may be added a step of selecting those of the aptamers of whichthe ability to bind to the target protein is not modified by thepresence of ethanol. The step of selecting these aptamers may be carriedout with a medium, including a buffer solution, comprising a finalethanol concentration of at least 1% (v/v), which includes at least1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%,7.5%, 8.0%, 9.0%, 9.5% and 10.0%.

Methods for Determining the Biological Activity of GLA-DomainCoagulation Proteins

Generally, the determination of the biological activity of theGLA-domain coagulation proteins is commonly carried out in a mannerknown to those skilled in the art according to either of two types oftests, respectively (i) a test for determining the anticoagulantactivity which takes advantage of the measurement of the property of thecoagulation protein under consideration of restoring the coagulantactivity of a plasma initially devoid of said coagulation protein, and(ii) a test for determining the actual enzymatic activity of thecoagulation protein under consideration, by measuring the conversion ofa chromogenic substrate. The general principles of these two types ofdetermination of the biological activity of a GLA-domain coagulationprotein are described below, as is their specific application to thebiological determination of the GLA-domain coagulation proteins ofinterest.

It is specified that the results of the measurements for determining thebiological activity of a GLA-domain coagulation protein are comparablefor a given test protein, regardless of the test used, insofar as theresults are expressed as a ratio between (i) the biological activity ofthe protein tested and (ii) the biological activity of an activereference protein.

According to the invention, the reference composition may consist of aplasma concentrate, preferentially a human plasma concentrate, or else acomposition comprising a recombinant GLA-domain coagulation protein.

Determination of the Biological Activity of a GLA-Domain CoagulationProtein by Measuring the Coagulant Activity

This first method consists in introducing the test coagulation factorinto a plasma initially devoid of this factor, then measuring thepercentage restoration of the anticoagulant activity relative to anormal plasma. Plasmas specifically depleted of each of the coagulationfactors are commercially available, for example through companies suchas Stago, American Diagnostica or Hyphen Biomed.

The activity of a plasma is characterized by a coagulation time, i.e. bya time which is necessary to form fibrin molecules which polymerize withone another and solidify the plasma. This modification of the mechanicalproperties of the plasma can be measured simply using coagulometerdevices. Coagulometer devices have a small magnetic bar, the movement ofwhich is stopped at the time of coagulation.

If the test coagulation factor is a procoagulant factor, the addition ofthis factor to a plasma initially depleted of this factor will cause adecrease in the coagulation time, which time will be close to that of anormal plasma. This type of method makes it possible to verify most ofthe functionalities of coagulation factors, such as: enzymatic activity,target and cofactor recognition, with the exception of the abilities tointeract with blood cells and the endothelial cells of the blood vesselwalls. This type of method gives an overall and averaged picture of theactivities of the GLA-domain coagulation factors to be tested.

Generally, the first method for determining the biological activity of aGLA-domain coagulation protein consists of a method which comprises thefollowing steps:

-   -   a) providing        -   (i) a test composition comprising a GLA-domain coagulation            protein,        -   (ii) a plurality of samples prepared from a reference            composition comprising a known amount of said biologically            active GLA-domain protein, each sample comprising a known            amount of said biologically active GLA-domain protein,            distinct from the amount of said protein contained in each            of the other samples,        -   (iii) a composition of plasma depleted of said GLA-domain            protein or a composition of plasma free of said GLA-domain            protein and, if necessary,        -   (iv) one or more purified cofactor(s) of said GLA-domain            protein,    -   b) beginning the assay by carrying out the following steps b1)        and b2):        -   b1) bringing (i) the test composition comprising a            GLA-domain coagulation protein into contact with (ii) the            composition of plasma depleted of said GLA-domain protein or            the composition of plasma free of said GLA-domain protein            and, if necessary, also with (iii) one or more purified            cofactor(s) of said GLA-domain protein,        -   b2) bringing (i) each sample of the reference composition            comprising a known amount of said biologically active            GLA-domain protein into contact with (ii) the composition of            plasma depleted of said GLA-domain protein or the            composition of plasma free of said GLA domain protein and,            if necessary, also with (iii) one or more purified            cofactor(s) of said GLA-domain protein,    -   c) determining:        -   c1) for the test composition, the time that elapses between            the beginning of step b1) and the moment at which the            mixture prepared in step b1) with the test composition is            coagulated, and        -   c2) for each sample of the reference composition, the time            that elapses between the beginning of step b2) and the            moment at which the mixture prepared in step b2) with the            reference composition is coagulated,    -   d) preparing a calibration curve, also called reference curve,        with the time measurements determined in step c2) as a function        of the known amount of said GLA-domain coagulation protein in        each sample,    -   e) determining the amount of said GLA-domain coagulation protein        contained in the test composition, by determining, on the        calibration curve prepared in step d), and on the basis of the        time determined in step c1), the deduced amount of said        GLA-domain coagulation protein in the test composition.        Determination of the Biological Activity of Factor II        (Prothrombin)

In step a)-(i), the test composition which comprises Factor II isprovided.

In step a)-(ii), a plurality of samples of the reference Factor IIcomposition are provided, for example in the form of samples ofsuccessive dilutions of a basic reference composition. It is, forexample, possible to use a first sample of the reference composition(100%), then successive dilutions to 50%, 25%, 12.5% and 6.2% of thereference composition.

In step a)-(iii), a Factor II-depleted plasma composition is provided,for example the composition sold by the company American DiagnosticaInc. (Stanford, USA) under the reference No. 202.

For carrying out the method, those skilled in the art may refer to thetest sold by the company Hyphen Biomed under the reference No. DP010K orthe test sold by the company Hyphen Biomed under the reference No.DP010A.

For carrying out the method, those skilled in the art may refer to thefollowing articles: Soulier et al. (1952, Sang, Vol. 23: 549-559),Favre-Gilly et al. (1967, Cah Med Lyonnais, Vol. 43(28): 2611-2668),Gjonaess et al. (Acta Obste Gynecol Scand, Vol. 54: 363-367) or Andrewet al. (1987, Blood, Vol. 70: 165-172).

Determination of the Biological Activity of Factor VII

In step a)-(i), the test composition which comprises Factor VII isprovided.

In step a)-(ii), a plurality of samples of the reference Factor VIIcomposition are provided, for example in the form of samples ofsuccessive dilutions of a basic reference composition. It is, forexample, possible to use a first sample of the reference composition(100%), then successive dilutions to 50%, 25%, 12.5% and 6.2% of thereference composition. A plasma concentrate sold by the NationalInstitute for Biological Standards and Control (NIBSC-United Kingdom)under the reference No. 97/592 can be used as reference composition.

In step a)-(iii), a Factor VII-depleted plasma composition is provided,for example the composition sold by the company American DiagnosticaInc. (Stanford, USA) under the reference No. 267.

For carrying out the method, those skilled in the art may refer to thetest sold by the company Hyphen Biomed under the reference No. DP030K orthe test sold by the company Hyphen Biomed under the reference No.DP030A.

For carrying out the method, those skilled in the art may refer to thefollowing articles: Soulier et al. (1952, Sang, Vol. 23: 549-559) orGjonaess et al. (Acta Obste Gynecol Scand, Vol. 54: 363-367).

Determination of the Biological Activity of Factor IX

In step a)-(i), the test composition which comprises Factor IX isprovided.

In step a)-(ii), a plurality of samples of the reference Factor IXcomposition is provided, for example in the form of samples ofsuccessive dilutions of a basic reference composition. It is, forexample, possible to use a first sample of the reference composition(100%), then successive dilutions to 50%, 25%, 12.5% and 6.2% of thereference composition. A plasma concentrate sold by the NationalInstitute for Biological Standards and Control (NIBSC-United Kingdom)under the reference No. 07/182 can be used as reference composition.

In step a)-(iii), a Factor IX-depleted plasma composition is provided,for example the composition sold by the company American DiagnosticaInc. (Stanford, USA) under the reference No. 269.

For carrying out the method, those skilled in the art may refer to thetest sold by the company Hyphen Biomed under the reference No. DP050K orthe test sold by the company Hyphen Biomed under the reference No.DP050A.

For carrying out the method, those skilled in the art may refer to thefollowing articles: Van Hylckama et al. (2000, Blood, Vol. 95(12):3678-3682), Taran et al. (1997, Biochemistry (Mosc.), Vol. 62(7):685-693), Orstavik et al. (1979, Br J Haematol, Vol. 42(2): 293-301),and also to the document available at the following internet address:“www.ncbi.nlm.nih.gov” (OMIM, Haemophilia B, FIX deficiency, +306900,+134540, +134510, +134520).

Determination of the Biological Activity of Factor X

In step a)-(i), the test composition which comprises Factor X isprovided.

In step a)-(ii), a plurality of samples of the reference Factor Xcomposition is provided, for example in the form of samples ofsuccessive dilutions of a basic reference composition. It is, forexample, possible to use a first sample of the reference composition(100%), then successive dilutions to 50%, 25%, 12.5% and 6.2% of thereference composition. A plasma concentrate as described in the HyphenBiomed references No. DP060K and DP060A can be used as referencecomposition.

In step a)-(iii), a Factor X-depleted plasma composition is provided,for example the composition sold by the company American DiagnosticaInc. (Stanford, USA).

For carrying out the method, those skilled in the art may refer to thetest sold by the company Hyphen Biomed under the reference No. DP060K orthe test sold by the company Hyphen Biomed under the reference No.DP060A.

For carrying out the method, those skilled in the art may refer to thefollowing articles: Favre-Gilly et al. (1967, Cah Med Lyonnais, Vol.43(28): 2611-2668) or Gjonaess et al. (Acta Obste Gynecol Scand, Vol.54: 363-367).

Determination of the Biological Activity of Protein C and of Protein S

The same method is applied for the determination of the biologicalactivity of protein C and of protein S.

Determination of the Biological Activity of a GLA-Domain CoagulationProtein by Measuring the Enzymatic Activity Using a DetectableSubstrate, for Example a Chromogenic or Fluorogenic Substrate

The basic principle of this second method is identical for all theGLA-domain coagulation factors. This second method consists in bringingthe factor to be characterized into contact with a chromogenic orfluorogenic substrate peptide, which mimics the peptide sequencenaturally recognized by said factor under consideration and which islysed by said factor on its natural target protein(s).

The release of the chromophore or of the fluorophore is measured using asuitable spectrometer. This method may require the addition of acofactor and, if necessary, the implementation of a step of activatingthe test factor. This second method makes it possible to measure theenzymatic functionalities of the GLA-domain coagulation protein testedwith the cofactors. It is a less overall method than the method formeasuring the coagulant activity previously described. However, thissecond method has the advantage of measuring the enzymatic activity ofthe GLA-domain coagulation protein of interest. Owing to a possibilityof cross reaction(s) with other enzymes having trypsin-like activity,this second method is used mainly for measuring the biological activityof factors that are prepurified, concentrated or present in a matrix notcontaining other enzymes in significant amount, compared with the amountof the coagulation factor of interest.

In step a)-(iii) a protein C-depleted plasma composition is provided,for example the composition sold by the company American DiagnosticaInc. (Stanford, USA) under the No. 249.

In step a)-(iii) a protein C-depleted plasma composition is provided,for example the composition sold by the company American DiagnosticaInc. (Stanford, USA), under the No. 253.

Generally, the second method for determining the biological activity ofa GLA-domain coagulation protein consists of a method which comprisesthe following steps:

-   -   a) providing        -   (i) a test composition comprising a GLA-domain coagulation            protein,        -   (ii) a plurality of samples prepared from a reference            composition comprising a known amount of said biologically            active GLA-domain protein, each sample comprising a known            amount of said biologically active GLA-domain protein,            distinct from the amount of said protein contained in each            of the other samples, and        -   (iii) a detectable substrate of said GLA-domain protein, or            a detectable substrate of a coagulation protein which is            activated, directly or indirectly, by said GLA-domain            protein,    -   b) beginning the assay by carrying out the following steps b1)        and b2):        -   b1) bringing (i) the test composition comprising a            GLA-domain coagulation protein into contact with (ii) a            detectable substrate of said GLA-domain protein or            with (iii) a detectable substrate of a coagulation protein            which is activated, directly or indirectly, by said            GLA-domain protein, in which case a composition comprising            one or more coagulation protein(s) which is (are) activated,            directly or indirectly, by said GLA-domain protein, and of            which the coagulation protein that is the last to be            activated consists of the coagulation protein capable of            converting said detectable substrate, is also added,        -   b2) bringing (i) each sample of the reference composition            comprising a known amount of said biologically active            GLA-domain protein into contact with (ii) a detectable            substrate of said GLA-domain protein or with (iii) a            detectable substrate of a coagulation protein which is            activated, directly or indirectly, by said GLA-domain            protein, in which case a composition comprising one or more            coagulation protein(s) which is (are) activated, directly or            indirectly, by said GLA-domain protein, and of which the            coagulation protein that is the last to be activated            consists of the coagulation protein capable of converting            said detectable substrate, is also added,    -   c) allowing the enzymatic conversion of said detectable        substrate to take place for a predetermined time starting,        respectively, from the beginning of step b1) and from the        beginning of step b2), and then stopping the enzymatic reaction        at the end of said predetermined time,    -   d) measuring the amount of said detectable substrate which has        been enzymatically converted:        -   d1) for each sample of the reference composition, and        -   d2) for the test composition,    -   e) preparing a calibration curve, also called reference curve,        with the measurements of the amount of said detectable substrate        which has been enzymatically converted, determined in step d1),        as a function of the known amount of said GLA-domain coagulation        protein in each sample of the reference composition,    -   f) determining the amount of said GLA-domain coagulation protein        contained in the test composition, by determining, on the        calibration curve prepared in step e), and on the basis of the        amount of said detectable substrate which was enzymatically        converted, determined in step d2), the deduced amount of said        GLA-domain coagulation protein in the test composition.

The term “detectable” substrate is intended to mean a substrate which isconverted, owing to the enzymatic reaction under consideration, into aconverted product that is detectable. Generally, a chromogenic orfluorogenic substrate is used.

Determination of the Biological Activity of Factor II (Prothrombin)

For carrying out this method, those skilled in the art can use thecolorimetric test sold by the company Hyphen Biomed under the referenceNo. 221605.

The standard curve can be prepared using the undiluted referencecomposition (200% prothrombin) and a series of diluted samples of thereference composition, for example samples diluted respectively to 160%,100% and 10% prothrombin, and also a prothrombin-free sample.

For carrying out this method, those skilled in the art may refer to thefollowing articles: Gomez et al. (2002, Clin Neurol Neurosurg, Vol.104(4): 285-288), Delahousse et al. (2002, Blood Coagulation &Fibinolysis, Vol. 13(5): 465-470), Neville et al. (2001, Haemostasis,Vol. 31: 211-217), Lane et al. (1996, Thromb Haemost, Vol. 76(5):651-662), Poort et al. (Blood, Vol. 88: 3698-3703), Rosen et al. (1999,ISTH, Abstract 269) or Stocker et al. (1996, Toxicon, Vol. 24(1): 81-89)or else to the documentation available at the following internetaddress: www.ncbi.nlm.nih.gov (OMIM, Coagulation Factor II, +176930).

Determination of the Biological Activity of Factor VII

For carrying out this method, those skilled in the art can use thecolorimetric test sold by the company Hyphen Biomed under the referenceNo. 221304.

The standard curve can be prepared using the undiluted referencecomposition (200% prothrombin) and a series of diluted samples of thereference composition, for example samples diluted respectively to 100%,10% and 1% Factor VII.

For carrying out this method, those skilled in the art may refer to thefollowing articles: Seligsohn et al. (1978, Blood, Vol. 52(5): 978-988),Avvisati et al. (1980, Br J Haematol, Vol. 45(2): 343-352), Poller etal. (1981, Br J Haematol, Vol. 49(1): 69-75), Van Diejien et al. (1982,Haemostasis, Vol. 12(3): 241-255), Clarke et al. (1992, FEBS Lett, Vol.298(2-3): 206-210), Ledwozyw et al. (1993, Arch Vet Pol, Vol. 33(1-2):123-127), Van Wersch et al. (1993, Int J Clin Lab Res, Vol. 23(4):221-224), Devies et al. (1997, Vol. 76(5): 405-408), Topper et al.(1998, Am J Vet Res, Vol. 59(5): 538-541) or Chang et al. (1999,Biochemistry, Vol. 28(34): 10940-10948).

Determination of the Biological Activity of Factor IX

For carrying out this method, those skilled in the art can use thecolorimetric test sold by the company Hyphen Biomed under the referenceNo. 221812.

The standard curve can be prepared using a series of diluted samples ofthe reference composition, for example samples comprising respectively afinal Factor IXa concentration of 1.4, 3.4, 6.8 and 13.5 mU/ml of FactorIXa.

For carrying out this method, those skilled in the art may refer to thefollowing articles: Taran et al. (1997, Biochemistry (Mosc.), Vol.62(7): 685-693), Wagenwoord et al. (1990, Haemostasis, Vol. 20(5):276-288) or the documentation available at the following

internet address: “www.ncbi.nlm.nih.gov” (OMIM, Haemophilia B, FIXdeficiency, +306900, +134540, +134510, +134520).

For carrying out this method, those skilled in the art can use thecolorimetric test sold by the company Hyphen Biomed under the referenceNo. 221802. 221605.

The standard curve can be prepared using the undiluted referencecomposition (200% Factor IXa) and a series of diluted samples of thereference composition, for example samples diluted respectively to 100%,50% and 20% Factor IXa.

For carrying out this method, those skilled in the art may refer to thefollowing articles: Van Hylckama et al. (2000, Blood, Vol. 95(12):3678-3682), Taran et al. (1997, Biochemistry (Mosc.), Vol. 62(7):685-693), Wagenwoord et al. (1990, Haemostasis, Vol. 20(5): 276-288),Parekh et al. (1978, Br J Haematol, Vol. 40(4): 643-655), Orstavik etal. (1979, Br J Haematol, Vol. 42(2): 293-301) or the documentationavailable at the following internet address: “www.ncbi.nlm.nih.gov”(OMIM, Haemophilia B, FIX deficiency, +306900, +134540, +134510,+134520).

Determination of the Biological Activity of Factor X

For carrying out this method, those skilled in the art can use the testsold by American Diagnostica Inc., denoted Actichrome® FX, under thereference No. 880.

The standard curve can be prepared using the undiluted referencecomposition (100% Factor Xa) and a series of diluted samples of thereference composition, for example samples diluted respectively to 75%,50%, 25%, 10% and 5% Factor IXa, and also a sample free of Factor X.

For carrying out this method, those skilled in the art may refer to thefollowing articles: Kisiel et al. (1976, Biochemistry, Vol. 15:4901-4906), Aurell et al. (1977, Thrombosis Research, Vol. 11: 595-605),Lindhout et al. (1978, Biochem Biophys Acta, Vol. 533: 327-341) orBergstrom et al. (1978, Thrombosis Research, Vol. 12: 531-547).

Determination of the Biological Activity of protein C

For carrying out this method, those skilled in the art can use the testsold by American Diagnostica Inc., denoted Actichrome® Protein C, underthe reference No. 836.

The standard curve can be prepared using the undiluted referencecomposition (100% protein C) and a series of diluted samples of thereference composition, for example samples diluted respectively to 75%,50%, 25%, 10% and 5% protein C, and also a protein C-free sample.

For carrying out this method, those skilled in the art may refer to thefollowing article: Francis et al. (1987, American journal of ClinicalPathology, Vol. 85(5): 619-625).

Table of sequences SEQ ID No. Type Designation 1 Nucleic acid 5′ regionof the aptamer 2 3′ region of the aptamer 3 Mapt-1 core sequence 4Mapt-1 aptamer 5 Aptamer not related to Mapt-1 6 to 34 Aptamers (“coresequences”) 35 Mapt-1.2.-CS aptamer 36 Mapt-1.2.-CSO aptamer 37Mapt-2-CS aptamer 38 Mapt-2.2.-CS aptamer 39 Mapt-2 aptamer

The present invention is also illustrated by the following examples.

EXAMPLES Example 1 Preparation of an Affinity Support

The affinity support was made from a solid support material consistingof a matrix onto which streptavidin (streptavidin-agarose—Novagen®) wasgrafted.

A volume of 1 ml of gel was introduced into a container consisting of acolumn (i.d. 11 mm). The gel was washed with purified water in order toremove the storage solvent.

The Gel Characteristics are:

-   -   Biotin adsorption capacity: ≧85 nanomol/ml of gel    -   Functional test: Capture >99% of biotinylated thrombin in 30        minutes at AT    -   Other tests: Protease-free, endo/exonuclease-free, RNase-free    -   Preservative: 100 mM sodium phosphate, pH 7.5, +NaN₃ 0.02.

The output of the packed column (gel bed height=1 cm) is connected to adetector of UV absorbance at 254 nm and a recording device.

The biotinylated anti-GLA nucleic aptamers of sequence SEQ ID No. 4,also called “Mapt-1” aptamers in the present description, aresolubilized in purified water at a final concentration of 0.5 mg/0.187ml, i.e. a final molar concentration of 0.1 mM. The solution of nucleicaptamers was activated at 95° C. according to the standard cycle, forimmobilizing the aptamers on the solid support material.

The solution of nucleic aptamers was previously diluted with 4.8 ml ofpurified water then 1.5 ml of concentrated buffer so as to obtain thefollowing formulation: 50 mM Tris, 50 mM NaCl, 4 mM MgCl₂, 10 mM CaCl₂,pH 7.5.

The absorbance detector is adjusted to 1 AUFS (Absorbance Unit FullScale) and the OD at 254 nm of this solution is recorded at 0.575 AU₂₅₄.

The solution of biotinylated nucleic aptamers is injected onto theprepacked streptavidin-agarose gel and recirculated with a peristalticpump at a flow rate of 2.5 ml/minute, i.e. a contact time on the gel of24 seconds (input/output I/O). Under these conditions, the OD at 254 nmstabilizes rapidly at 0.05 AU₂₅₄, which is 91% of the theoreticalcoupling, i.e. 0.455 mg of nucleic aptamers per milliliter of gel.

Washing with a 2M NaCl buffer is carried out, in order to remove thenucleic aptamers that were not bound specifically to the streptavidinmolecules grafted onto the solid support material.

Example 2 Purification of a Recombinant GLA-Domain Protein (TransgenicHuman Factor IX)

An affinity support of the type described in example 1 was used topurify recombinant human factor IX produced in the milk of pigstransgenic for human Factor IX.

A. Protocol for Purifying by Affinity Chromatography

The characteristics for implementing the step of affinity chromatographyof the starting sample on an affinity support on which the Mapt-1anti-GLA aptamers are immobilized are described in the tables below.

Step 1: Clarification

Clarification with citrate to obtain clarified milk at pH 7.5 at a finalconcentration of 0.25M of citrate buffer.

Step 2: MEP hypercel

SM: clarified milk (IBF 25-10 ml of gel)

FIX loading: 243 IU/ml of gel

Equilibration buffer: 0.25M citrate, pH 7.5

Elution buffer: water

Step 3: Dialysis

SM: MEP eluate

Dialysis buffer: 50 mM Tris-50 mM NaCl, pH 7.5

Step 4: MAPT-1 (3 run)

SM: Dialyzed MEP eluate (IBF 1.1-1 ml of gel)

FIX loading: 230 IU/ml of gel

Equilibration buffer: 50 mM Tris-50 mM NaCl-4 mM MgCl₂-10 mM CaCl₂, pH7.5

Elution buffer: 20 mM Tris-10 mM EDTA, pH 7.5

Regeneration buffer: 20 mM Tris-1M NaCl-5% PG, pH 7.5

B. Characteristic of the Raw Material (RM)

The raw material used consists of untreated milk from a pig transgenicfor human Factor IX.

C. Monitoring of the Process

C.1. Step 1: Clarification

C.1.1.—Monitoring of Clarification

-   -   Thawing of E0 at 37° C.    -   Mixture with a very milky appearance: 45 g (⅔) pig's milk+25 g        (⅓) citrate buffer concentrated to 0.75M    -   Gentle stirring for 30 min at ambient temperature    -   Centrifugation for 30 min, 15° C., 5000 g    -   Pellet: small and white containing pig hairs and impurities    -   Supernatant: two phases, the upper solid phase creamy and white        constituting the fatty substances and the yellowish phase        representing the clarified milk (E1    -   Clarified milk recovered with pump    -   Deep filtration of the clarified milk with a Cuno BioCap 25        Filter    -   Deep freezing at −80° C. of the clarified filtered milk (E2).        C.1.2—Results in Total Proteins, in FIX:C, FIX:Ag and FIX:Am

The results are shown in tables 1 and 2 below.

TABLE 1 Coagulation Total proteins Q R Q R Purification Weight [FIX:C]FIX:C FIX:C [Prot] Prot. Prot. AS Increased Step Code (g) (IU/ml) (IU)(%) (g/l) (g) (%) (IU/mg) Purity purity Untreated milk E0 45.0 57.2 2574100.0 103.2 644 100.0 0.6 0.2 / Clarified milk E1 65.3 36.0 2350 91.337.5 2451 52.8 1.0 0.4 1.7 before filtration Clarified milk E2 63.8 38.22438 94.7 34.7 2214 47.7 1.1 0.5 2.0

TABLE 2 Amidolytic Antigenic Q R Q R Ratio [FIX:Am] FIX:Am FIX:Am[FIX:Ag] FIX:Ag FIX:Ag [C]/ [Am]/ Code (IU/ml) (IU) (%) (IU/ml) (IU) (%)[Ag] [Ag] E0 40 1798 100 147 6615.0 100.0 0.4 0.3 E1 23 1510 84 1147443.1 112.5 0.3 0.2 E2 22 1426 79 95 6062.0 91.6 0.4 0.2C.2. Step 2: MEP HypercelC.2.1.—Characteristics of the Chromatography Support

-   -   Column: IBF 25, 2.5 cm high    -   Gel: MEP 10 ml of gel, batch 200920/G206-04    -   No. of use: 2^(nd) use    -   Regeneration of the gel before use: 1 CV 1M NaOH; 2 CV 2M NaCl;        4 CV water

C.3.2—Preparation of the Starting Material (SM)

-   -   Thawing of E2 at 37° C.    -   Reference: 1 IU of FIX=4 μg/ml    -   Injected volume (g): 63.81    -   FIX loading in IU per ml of gel (IU/ml gel): 243 IU/ml of gel    -   FIX loading in μg per ml of gel (μg/ml gel): 972 μg/ml of gel        C.2.3—Characteristics of Chromatography Buffers    -   Equilibration buffer: 0.25M citrate, pH 7.5, 612 mOsmol/kg, 31.9        mS/cm        MEP elution buffer: water        C.2.4—Monitoring of Chromatography Steps

TABLE 3 Flow rate Weight Peak Osmolarity Conductiv. Code Step ml/min gAU pH mosmol/kg mS/cm Observations E2 SM 0.85 63.81 NA 7.43 231 31.9 NAE3 NR 0.85 139.61 2 7.45 719 32 Saturation E4 E MEP 1 51.68 2 7.81 1217.11 Saturation Regeneration of the gel before use: 10CV 1M NaOH; 4CV 2MNacl; 10CV water; 10CV 20% ethanol.C.2.5. Results in Total Proteins

TABLE 4 Coagulation Total proteins Q R Q R Purification Weight [FIX:C]FIX:C FIX:C [Prot] Prot. Prot. AS Increased Steps Code (g) (IU/ml) (%)(%) (g/l) (g) (%) (IU/mg) Purity purity Clarified milk E2 63.8 38.2 2438100.0 34.7 2214.2 100.0 1.1 0.5 / Nonadsorbed E3 139.6 3.1 433 17.8 8.41175.4 53.1 0.4 0.2 0.3 Elution MEP E4 51.7 20.3 1049 43.0 3.6 186.6 8.45.6 2.5 5.1 MEP eluate dialyzed E5 51.1 14.5 741 30.4 3.3 169.1 7.6 4.41.9 4.0

TABLE 5 Amidolytic Antigenic Q R Q R Ratio [FIX:Am] FIX:Am FIX:Am[FIX:Ag] FIX:Ag FIX:Ag [C]/ [Am]/ Code (IU/ml) (IU) (%) (IU/ml) (IU) (%)[Ag] [Ag] E2 22 1426 100 95 6062.0 100.0 0.4 0.2 E3 1 148 10 ND ND ND NDND E4 9 484 34 47 2429.0 40.1 0.4 0.2 E5 9 455 32 45 2298.6 37.9 0.3 0.2C.3. Step 3: Dialysis

Dialysis of E4 (50 g) in 2 baths of 1 L, all night with stirring at 4°C.

TABLE 6 Weight Osmolarity Conductivity Samples Fractions g pH mosmol/kgmS/cm E4 before dialysis 49.18 7.81 121 7.11 E5 after dialysis 51.087.81 149 7.18 Dialysis buffer 7.50 157 7.52 50 mM Tris - 50 mM NaClC.4.: Chromatography on Affinity Support with Anti-GLA Aptamers (Mapt-1)

Three independent purification tests for transgenic human Factor IXpre-purified as described above were carried out. The operatingconditions and the results of each test are specified below.

C.4.1—Characteristics of the Chromatography Support

-   -   Column: IBF 1.1, 0.9 cm high    -   Gel: MAPT-1, 1 ml of gel    -   No. of use: 2^(nd) use    -   C.4.2—Preparation of the Starting Material (SM)    -   Adjustment of pH and addition of 4 mM MgCl₂ and 10 mM CaCl₂        final concentration, to E5    -   Aliquoting of E5 (48.17 g), aliquoted as 3×16 g, two of which        are stored at −80° C. for the following tests    -   Reference: 1 IU of FIX=4 μg/ml    -   Injected volume (g): 16.14    -   FIX loading in IU per ml of gel (IU/ml gel): 234 IU/ml of gel    -   FIX loading in μg per ml of gel (μg/ml gel): 936 μg/ml of gel        C.4.3. Characteristics of Chromatography Buffers    -   Equilibration buffer: 50 mM Tris, 50 mM NaCl, 10 mM CaCl₂, 4 mM        MgCl₂, pH 7.51, 218 mOsmol/kg, 10.77 mS/cm    -   Elution buffer 1: 20 mM Tris, 10 mM EDTA, pH 7.53, 56 mOsmol/kg,        2.64 mS/cm    -   Elution buffer 2 (regeneration buffer): 20 mM Tris, 1M NaCl, 50%        propylene glycol, pH 7.52, 18.27 mS/cm.        C.4.4. Monitoring of Chromatography Steps        Test 1    -   Column equilibrated at pH 7.34 and 211 mOsm/kg    -   FIX loading: 234 IU/ml of gel.

TABLE 7 Flow rate Weight Peak Osmolarity Conductiv. Samples Fractionsml/min g AU pH mosmol/kg mS/cm Observations E5 SM 0.6 16.14 na 7.51 1818.18 na E6 NR 0.6 29.56 0.5 7.44 193 9.03 saturation E7 E1 MAPT-1 0.66.68 0.02 6.40 99 na decrease in pH E8 E2 MAPT-1 0.6 6.66 0.1 7.41 na nanaTest 2

-   -   Column equilibrated at pH 7.39 and 220 mOsm/kg, 3^(rd) use of        the gel    -   FIX loading: 236 IU/ml of gel.

TABLE 8 Fractions Flow rate Weight Peak Osmolarity 315 256 ml/min g AUpH mosmol/kg Observations SM 0.1 13.50 na 7.42 179 Na NR 0.1 20.29 0.57.36 197 saturation E1 MAPT-1 0.1 2.84 0.02 7.06 187 decrease in pH E2MAPT-1 0.1 3.77 0.1 6.39 na decrease in pHTest 3

-   -   Column equilibrated at pH 7.45 and 219 mOsm/kg, 5^(th) use of        the gel    -   FIX loading: 210 IU/ml of gel.

TABLE 9 Fractions Flow rate Weight Peak Osmolarity 315 258 ml/min g AUpH mosmol/kg Observations SM 0.6 12.71 na 7.47 180 na NR 0.6 23.79 0.57.46 206 saturation E1 MAPT-1 0.6 2.67 0.02 6.94 168 decrease in pH E2MAPT-1 0.6 4.20 0.1 7.36 na naC.4.5. Results in Total Proteins, in FIX:C, FIX:Ag and FIX:Am

The assays of total proteins were carried out by the Tebu Biolaboratory. FIX:C levels were determined by LBA and FIX:Ag and FIX:Amlevels by LIB.

C.4.5.1 Affinity Chromatography Results, Test 1

TABLE 10 Protein Coag R R Q Total Purification Weight [FIX:C] Q FIX:C[Total Total prot. AS Increased Step Code (g) (IU/ml) FIX:C (%) prot.]prot. (%) (IU/mg) Purity purity Injection E5 16.1 14.5 234 100.0 3.31053.4 100.0 4.4 1.9 / Nonadsorbed E6 29.6 8.9 263 112.4 1.360 40.2 75.36.5 2.9 1.5 Elution 1 E7 6.7 0.50 3.3 1.4 0.015 0.1 0.2 33.3 14.7 7.6

TABLE 11 Am Ag Q R Q R Ratio [FIX:Am] FIX:Am FIX:Am [FIX:Ag] FIX:AgFIX:Ag [C]/ [Am]/ Code (IU/ml) (IU) (%) (IU/ml) (IU) (%) [Ag] [Ag] E5 9143.6 100.0 45 726.3 100.0 0.3 0.2 E6 2.7 78.6 54.7 26 768.6 105.8 0.30.1 E7 0.2 1.1 0.7 0.238 1.6 0.2 2.1 0.74.5.2. Affinity Chromatography Results, Test 2

TABLE 12 Protein Coag R R Q Total Purification Weight [FIX:C] Q FIX:C[Total Total prot. AS Increased Step Code (g) (IU/ml) FIX:C (%) prot.]prot. (%) (IU/mg) Purity purity Injection SM 13.5 17.5 236 100.0 3.11042.0 100.0 5.6 2.5 / Nonadsorbed NR 20.3 8.7 177 74.7 1.940 39.4 93.84.5 2.0 0.8 Elution 1 E1 2.8 0.32 0.91 0.4 0.007 0.0 0.0 45.7 20.1 8.1

TABLE 13 Am Ag Q R Q R Ratio [FIX:Am] FIX:Am FIX:Am [FIX:Ag] FIX:AgFIX:Ag [C]/ [Am]/ Code (IU/ml) (IU) (%) (IU/ml) (IU) (%) [Ag] [Ag] SM9.4 127.4 100.0 45.0 607.5 100.0 0.4 0.2 NR 5 100.4 78.8 25.0 507.3 83.50.3 0.2 E1 0.1 0.4 0.3 0.2 0.6 0.1 1.6 0.7C.4.5.3. Affinity Chromatography Results, Test 3

TABLE 14 Protein Coag R R Q Total Purification Weight [FIX:C] Q FIX:C[Total Total prot. AS Increased Step Code (g) (IU/ml) FIX:C (%) prot.]prot. (%) (IU/mg) Purity purity Injection SM 12.7 16.5 210 100.0 4.43056.3 100.0 3.7 1.6 / Nonadsorbed NR 23.8 8.0 190 90.8 2.130 50.7 90.03.8 1.7 1.0 Elution 1 E1 2.7 1.8 4.8 2.3 0.011 0.0 0.1 163.6 72.1 43.9

TABLE 15 Am Ag Q R Q R Ratio [FIX:Am] FIX:Am FIX:Am [FIX:Ag] FIX:AgFIX:Ag [Am]/ [C]/ Code (IU/ml) (IU) (%) (IU/ml) (IU) (%) [C]/[Ag] [Ag][Am] SM 9.4 119.1 100.0 47 597.4 100.0 0.4 0.2 1.8 NR 4.4 105.6 88.7 23547.2 91.6 0.3 0.2 1.8 E1 0.5 1.4 1.2 0.80 2.1 0.4 2.3 0.7 3.3 E2 221426 79 95 6062.0 91.6 0.4 0.2 1.7

Example 3 Purification of a GLA-Domain Protein with a CorrectlyGamma-Carboxylated GLA Domain

In example 3, tests were carried out in particular for purifying a GLAprotein with a correctly gamma-carboxylated GLA domain, from a startingsample comprising a mixture of different forms of the same GLA-domainprotein, respectively forms in which the GLA domain is correctlygamma-carboxylated and forms in which the GLA domain is incorrectlygamma-carboxylated.

More specifically, tests were carried out for purifying recombinantFactor IX produced in the milk of pigs transgenic for human Factor IX.The milk of the transgenic pigs comprises a mixture of (i) activetransgenic recombinant human Factor IX with a correctlygamma-carboxylated GLA domain and (ii) inactive transgenic recombinanthuman Factor IX with an incorrectly gamma-carboxylated GLA domain.

The results of these tests are given below.

4.1. Selective Binding of Mapt-1 to GLA Proteins with a CorrectlyGamma-Carboxylated GLA Domain

4.1.1. Experimental Conditions

a) Binding measurement: Biacore T100 Apparatus

-   -   Chip: the biotinylated Mapt-1 aptamer was immobilized on a        streptavidin surface (chip SA, GE) at 4326 RU on the active flow        cell No. 3 (FC3). A control nucleic aptamer is immobilized at        4069 RU on the flow cell No. 1 (FC1). The injected sample runs        over FC3 and FC1 in order to completely extract the background        noise due to nonspecific interactions.    -   Run buffer and dilution buffer for samples: 50 mM Tris/50 mM        NaCl/10 mM CaCl₂/4 mM MgCl₂/pH=7.4.    -   Flow rate: 30 μl/min, injection for 60 s, dissociation for 120 s    -   Signal: Fc3 signal corrected for the flow cell No. 1 signal    -   Regeneration: 10 mM EDTA in water for injection.

More specifically, a first solid support (FC3) was produced, on whichmolecules of the nucleic aptamer of the invention having the sequenceSEQ ID No. 4, also known here as “Mapt-1”, were immobilized. Beforebeing bound to the solid support, the 5′ end of the Mapt-1 aptamer waschemically coupled to a spacer chain consisting of one molecule of PEG(C18). Then, the free end of the spacer chain, opposite to the endcoupled to the aptamer, was coupled to a biotin molecule.

A second solid support (FC1) was also produced, on which molecules of anaptamer not related to Mapt-1, having the sequence SEQ ID No. 5, whichdoes not bind to GLA-domain proteins, were immobilized.

A solid support containing immobilized molecules of streptavidin isavailable (series S sensor chip SA, GE).

Then, the above solid support was brought into contact with the aboveaptamer compounds in order to immobilize the nucleic acids having thesequences SEQ ID No. 4 and SEQ ID No. 5, by noncovalent associationbetween the streptavidin molecules of the support and the biotinmolecules of the aptamer compounds.

The Mapt-1 aptamer is thus immobilized with an immobilization rate of4326 RU (1 RU corresponds approximately to 1 pg of product immobilizedper mm²).

Transgenic recombinant human FIX produced in pigs and prepurified bychromatography on an MEP HyperCel® support was diluted in run buffer (50mM Tris, 50 mM NaCl, 10 mM CaCl₂, 4 mM MgCl₂, pH 7.4).

The sample was injected sequentially onto (i) the FC1 chip containingthe non-related aptamer immobilized by a biotin-streptavidin interactionand (ii) the FC3 chip (solid support) containing the Mapt-1 aptamerimmobilized by a biotin-streptavidin interaction. Controls are obtainedby injecting blanks containing only run buffer. All the injections werecarried out with a flow rate of 30 μl/min for 60 sec. After theinjection, run buffer was injected onto the chip at an identical flowrate for 120 sec. Elution buffer (10 mM EDTA in water for injection) wasthen injected for 30 sec with a flow rate of 30 μl/min in order touncouple the substances bound to the aptamer. The chip makes it possibleto study in real time the formation and the breaking of interactionsbetween FIXtg and the immobilized aptamer through surface plasmonresonance (SPR). Binding to the immobilized aptamer generates anincrease in the signal expressed in resonance units (RU) recorded by theapparatus (FIG. 3). These analyses are carried out with the Biacore T100SPR apparatus (GE). The modeling of the recorded interactions is carriedout by means of the Biaevaluation software (GE).

The signal obtained with the FC1 chip is subtracted from the signalobtained with the FC3 chip in order to eliminate the background noisedue to nonspecific interactions.

b) Measurement of Specific Activity: Ratio of Amidolytic Activity OverAntigen Level

-   -   Amidolytic activity is measured using the Biophen FIX kit        according to the supplier's instructions.    -   The antigen level is measured using the Stago Asserachrom®        VII:Ag kit according to the supplier's instructions.        c) Samples:    -   Benefix® recombinant FIX (Nonacog alpha, Wyeth) with        γ-carboxylations according to the manufacturer's data.    -   FIX-TG purified from transgenic pig's milk (milk K97, supplied        by GTC) by various chromatography steps (assay 315138) in order        to obtain more than 95% purity. The γ-carboxylation of the light        chain was analyzed by LC-MS after activation and digestion with        PNGase F. The MS spectra showed different degrees of        γ-carboxylation on the light chain ranging from 1 to 7. The        normal level of γ-carboxylation is 12.        4.1.2. Results

The results are shown in FIG. 1.

The biacore binding measurements show that the Mapt-1 aptamer does notbind to the purified FIX-Tg sample characterized by an incomplete degreeof γ-carboxylation and a specific activity of 0.2. These results suggesta selectivity of Mapt-1 for the correctly γ-carboxylated forms.

4.2. Capacity of an Affinity Support According to the Invention toSelectively Retain Human Factor IX of which the GLA Domain is CorrectlyGamma-Carboxylated

Milk from a pig transgenic for human Factor IX was subjected to a methodsimilar to that described in example 2, comprising the following steps:

-   -   a) clarifying with citrate,    -   b) running the clarified milk on an MEP Hypercel® chromatography        support,    -   c) eluting the retained fraction and dialyzing the eluate        against a 50 mM Tris, 50 mM NaCl buffer, at pH 7.5, then        adjusting with 10 mM CaCl₂ and 4 mM MgCl₂,    -   d) running the eluate obtained at the end of step c) on an        affinity support on which anti-GLA aptamers are immobilized, in        this case the Mapt-1 aptamer.

Separately, as a control test, plasma factor IX was run on an affinitysupport on which anti-GLA aptamers are immobilized, in this case theMapt-1 aptamer.

4.2.1. Experimental Conditions

4.2.1.1. Chromatography Conditions

a) Step 1: Clarification

Clarification with citrate to obtain clarified milk at pH 7.5 at a finalcitrate buffer concentration of 0.25M

b) Step 2: Running on MEP Hypercel®

-   -   SM: clarified milk (IBF 25-10 ml of gel)    -   FIX loading: 243 IU/ml of gel    -   Equilibration buffer: 0.25M citrate, pH 7.5    -   Elution buffer: water.        c) Step 3: Dialysis    -   SM: MEP eluate    -   Dialysis buffer: 50 mM Tris-50 mM NaCl, pH 7.5.        d) Step 4: MAPT-1 (3 runs)    -   SM: Dialysed MEP eluate (IBF 1.1-1 ml of gel) adjusted for MgCl₂        and CaCl₂ so as to obtain the respective concentrations: 10 mM        and 4 mM    -   FIX loading: 230 IU/ml of gel    -   Equilibration buffer: 50 mM Tris-50 mM NaCl-4 mM MgCl₂-10 mM        CaCl₂, pH 7.5    -   Elution buffer: 20 mM Tris-10 mM EDTA, pH 7.5    -   Regeneration buffer: 20 mM Tris-1M NaCl-5% PG, pH 7.5.        4.2.1.2. Measurement of Specific Activity: Ratio of Amidolytic        Activity Over Antigen Level or of Coagulant Activity Over        Antigen Level    -   Amidolytic activity is measured using the Biophen FIX kit        according to the supplier's recommendations.    -   Coagulant activity is measured by a chronometric method that        consists in measuring the coagulation time in the presence of        cephalin and kaolin, of a system in which all the factors are        present in excess except for Factor IX (Factor IX deficient        plasma, Stago). The FIX is provided by the dilution of the        standard or of the sample. The analyses are done on an        auto-analyzer: type BCT and the data is processed by        “hemostasis” software for calculating the activity and the        confidence interval.    -   Antigen level is measured using the Stago Asserachrom® VII:Ag        kit according to the supplier's instructions.        4.2.1.3. Binding Measurement: Biacore T100 Apparatus    -   Chip: Mapt-1 immobilized on a streptavidin surface (chip SA, GE)        at 5073 RU on the active flow cell No. 2 (FC2). A non-relevant        aptamer is immobilized at 4959 RU on the flow cell No. 1. The        injected sample runs over FC2 and FC1 in order to completely        subtract the background noise due to nonspecific interactions.    -   Buffer for running and dilution of samples: 50 mM Tris/10 mM        CaCl₂/pH=7.4

Flow rate: 30 μl/min injection for 100 s, dissociation for 200 s

-   -   Signal: FC2 signal corrected for the flow cell No. 1 signal    -   Regeneration: 10 mM EDTA in PBS buffer, pH 7.4.        4.2.2. Results        4.2.2.1. Chromatography

A chromatogram was produced and also an SDS-PAGE gel electrophoresis wascarried out with Coomassie blue staining. The results are shown in FIG.2.

The analysis of the chromatogram of the Mapt-1 step shows first that thevast majority of the injected sample is not retained and, second, a veryweak elution peak that may correspond to minority species of FIX-TG.

As shown in the image of FIG. 3, the SDS-PAGE gel analysis of theeluates does not enable the visualization of possibly purified FIX,which is coherent with the quantity related to the observed peak height.

On the other hand, when an SDS-PAGE electrophoresis gel is prepared andthen stained with silver nitrate, protein bands of purified transgenicrecombinant human FIX, which has been shown to possess a very highbiological activity, are clearly visualized in the eluate of thefraction retained on the Mapt-1 affinity support (see the SDS-PAGE gelimage shown in FIGS. 3A and 3B).

4.2.2.2. Specific Activity of Factor IX Found in the Various Fractions

Table 19 below presents the results of biological activity of Factor IXin the starting product and in the various fractions obtained during theaffinity chromatography on the Mapt-1 affinity support.

TABLE 16 Test 1 Test 2 Test 3 Plasma controls Sample Test No. 09315255Test No. 09315256 Test No. 09315258 Test No. 09315257 Analysis [C]/[Ag][Am]/[Ag] [C]/[Ag] [Am]/[Ag] [C]/[Ag] [Am]/[Ag] [C]/[Ag] [Am]/[Ag] Start0.3 0.2 0.4 0.2 0.4 0.2 1.1 1.3 Nonabsorbed 0.3 0.1 0.3 0.2 0.3 0.2 1.00.7 Mapt eluate 2.1 0.7 1.6 0.7 2.1 0.7 1.2 1.74.2.2.3 Binding Test on Biacore® for Various Forms of Human Factor IX

The results are shown on the graph in FIG. 4.

The binding measurement results represented in FIG. 5 show that theFIX-TGs obtained in the weak elution peak are indeed characterized by asignificant affinity to Mapt-1, contrary to the majority of other formsof FIX-TG represented by the sample resulting from the purificationprocess.

In example 3, it is shown that the Mapt-1 affinity support possesses aselectivity of binding for the species of GLA-domain proteins with thehighest biological activity, in the case in point the most activespecies of transgenic recombinant human Factor IX.

Thus, in addition to enabling selective enrichment in Factor IX from thestarting sample, the Mapt-1 affinity support makes it possible toincrease the biological activity/Factor IX quantity ratio.

Example 4 Purification of Various GLA-Domain Proteins with an AffinitySupport on which Anti-GLA Aptamers are Immobilized

In example 4, it was shown that an affinity support on which anti-GLAaptamers are immobilized is successfully used for purifying a variety ofGLA-domain proteins.

More specifically, it was shown in example 4 that an affinity support onwhich Mapt-1 anti-GLA aptamers are immobilized selectively retainsFactor VII, Factor IX and Factor X.

5.1.1. Experimental Conditions

Binding measurement: Biacore T100 apparatus

-   -   Chip: Mapt-1 immobilized on a streptavidin surface (Chip SA, GE)        at 3596 RU on the active flow cell No. 2 (FC2). Buffer for        running and diluting samples: 50 mM Tris/50 mM NaCl/10 mM        CaCl₂/4 mM MgCl₂/pH=7.4    -   Flow rate: 30 μl/min injection for 60 s, dissociation for 120 s    -   Signal: FC2 signal corrected for the flow cell No. 1 signal        which is the blank    -   Regeneration: 10 mM EDTA in 50 mM Tris, pH=7.4.        5.1.2. Results

The results are given in FIG. 5.

FIG. 6 shows that the Mapt-1 affinity support selectively retains avariety of GLA-domain proteins, in the case in point a variety of humanGLA-domain coagulation proteins such as Factor VII, Factor IX and FactorX.

Example 5 Capture of Nucleic Acid Aptamers According to the Invention byHuman Factor IX Immobilized on a Support

A solid support on which purified recombinant human Factor IX moleculeswere immobilized was produced. A purified preparation of recombinanthuman Factor IX sold under the name Benefix® by the company Wyeth wasused.

Human Factor IX was immobilized on carboxymethyl dextran activated withNHS-EDC and which binds to the free amines present on FIX.

The human recombinant Factor IX is thus immobilized with animmobilization rate of 3153 RU (1 RU corresponds approximately to 1 pgof product immobilized per mm²).

Nucleic aptamers of the invention (purity: 99%), respectively theaptamers having the sequences SEQ ID Nos. 3 and 6 to 35, were diluted inrun buffer (50 mM Tris, 10 mM CaCl₂, 4 mM MgCl₂, pH 7.5) in order toobtain as many aptamer samples as distinct aptamers to be tested.

Each sample was injected sequentially onto the same chip (solid support)containing the immobilized human recombinant FIX. Controls are obtainedby injecting blanks containing only run buffer. All the injections werecarried out with a flow rate of 30 μl/min for 60 sec; after theinjection, run buffer was injected onto the chip at an identical flowrate for 120 sec.

Elution buffer (10 mM EDTA) was then injected for 60 sec with a flowrate of 30 μl/min to uncouple the aptamer from the immobilized humanFIX.

The chip makes it possible to observe in real time the formation and thedisruption of interactions between the immobilized human recombinant FIXand each of the aptamers, having the sequences SEQ ID Nos. 3 and 6 to35, tested, through surface plasmon resonance (SPR). Binding to theimmobilized recombinant human FIX generates an increase in signalexpressed in resonance units (RU) recorded by the apparatus (FIG. 6).These analyses are carried out with the Biacore T100 SPR apparatus (GE).The modeling of the recorded interactions is carried out by means of theBiaevaluation software (GE).

The results obtained show that all the nucleic aptamers tested bind withsignificant affinity to recombinant human plasma Factor IX.

The results of FIG. 6 also show that the 30 aptamers tested can beclassified in four main groups, according to their level of affinity forhuman Factor IX.

The very high affinity for human Factor IX of the aptamers classified ingroup 4 as shown in FIG. 6 may in particular be noted. Out of the 30aptamers tested, the aptamer with the highest affinity for human FactorIX, which is designated Mapt-1.2, consists of the aptamer having thesequence SEQ ID No. 35 [Mapt-1.2-CS].

Example 6 Capture of Nucleic Acid Aptamers According to the Invention byHuman Factor IX Immobilized on a Support

A solid support on which purified recombinant human Factor IX moleculeswere immobilized was produced. A purified preparation of recombinanthuman Factor IX sold under the name Benefix® by the company Wyeth wasused.

Human Factor IX was immobilized on carboxymethyl dextran activated withNHS-EDC and which binds to the free amines present on FIX.

The recombinant human Factor IX is thus immobilized with animmobilization rate of 3153 RU (1 RU corresponds approximately to 1 pgof product immobilized per mm²).

Nucleic aptamers of the invention (purity: 99%), respectively theaptamers having the sequences SEQ ID No. 35 [Mapt-1.2-CS] and SEQ ID No.36 [Mapt-1.2-CSO], were diluted in run buffer (50 mM Tris, 10 mM CaCl₂,4 mM MgCl₂, pH 7.5) in order to obtain as many aptamer samples asdistinct aptamers to be tested.

The Mapt-1.2.-CSO aptamer having the sequence SEQ ID No. 36 is derivedfrom the nucleic acid with the structure: 5′-SEQ ID No. 1-SEQ ID No.35-SEQ ID No. 2 that is 80 nucleotides in length. More specifically, theMapt-1.2.-CSO aptamer having the sequence SEQ ID No. 36 consists of thenucleic acid ranging from the nucleotide in position 10 and terminatedwith the nucleotide in position 49 of the nucleic acid with thestructure: 5′-SEQ ID No. 1-SEQ ID No. 35-SEQ ID No. 2.

Each sample was injected sequentially onto the same chip (solid support)containing the immobilized recombinant human FIX. Controls are obtainedby injecting blanks containing only run buffer. All the injections werecarried out with a flow rate of 30 μl/min for 60 sec; after theinjection, run buffer was injected onto the chip at an identical flowrate for 120 sec.

Elution buffer (10 mM EDTA) was then injected for 60 sec with a flowrate of 30 μl/min to uncouple the aptamer from the immobilized humanFIX.

The chip makes it possible to study in real time the formation and thedisruption of interactions between the immobilized recombinant human FIXand each of the aptamers, having the sequences SEQ ID No. 35[Mapt-1.2-CS] and SEQ ID No. 36 [Mapt-1.2-CSO] tested, through surfaceplasmon resonance (SPR). Binding to the immobilized recombinant humanFIX generates an increase in the signal expressed in resonance units(RU) recorded by the apparatus (FIG. 7). These analyses are carried outwith the Biacore T100 SPR apparatus (GE). The modeling of the recordedinteractions is carried out by means of the Biaevaluation software (GE).

The results obtained show that the two nucleic aptamers tested bind withsignificant affinity to recombinant human plasma Factor IX.

The results of FIG. 7 also show that, of the two aptamers tested,Mapt-1.2.-CSO (SEQ ID No. 36) exhibits a level of affinity for humanFactor IX that is significantly higher than the level of affinity of theMapt-1.2.-CS aptamer (SEQ ID No. 35).

The results of FIG. 7 also show the excellent stability of the bindingof the Mapt-1.2.-CSO aptamer (SEQ ID No. 36) to Factor IX.

It is recalled that the Mapt-1.2.-CS aptamer (SEQ ID No. 35) was theaptamer exhibiting the highest level of affinity of the 30 aptamerstested in example 5.

Example 7 Preparation of an Affinity Support

The affinity support was made from a solid support material consistingof a matrix on which streptavidin (streptavidin-agarose—Novagen®) wasgrafted.

A volume of 1 ml of gel was introduced into a container consisting of acolumn (i.d. 11 mm). The gel was washed with purified water, to removethe storage solvent.

The Gel Characteristics are:

Biotin adsorption capacity: ≧85 nanomol/ml of gel

-   -   Functional test: Capture >99% of biotinylated thrombin in 30        minutes at AT    -   Other tests: Protease-free, endo/exonuclease-free, RNase-free    -   Preservative: 100 mM sodium phosphate, pH 7.5, +NaN₃ 0.02.

The output of the packed column (gel bed height=1 cm) is connected to anabsorbance detector fitted with a UV filter at 254 nm and a recordingdevice.

Biotinylated anti-human FIX nucleic aptamers comprising the nucleic acidhaving the sequence SEQ ID No. 4 [Mapt-1-WS] are solubilized in purifiedwater at a final concentration of 0.5 mg/0.187 ml, i.e. a final molarconcentration of 0.1 mM. The solution of nucleic aptamers was activatedat 95° C. according to the standard cycle, for immobilizing the aptamerson the solid support material.

The solution of nucleic aptamers was diluted beforehand with 4.8 ml ofpurified water then 1.5 ml of Mg⁺⁺ buffer (concentrated 5×).

The absorbance detector is adjusted to 1 AUFS (Absorbance Unit FullScale) and the OD at 254 nm of this solution is recorded at 0.575 AU₂₅₄.

The solution of biotinylated nucleic aptamers is injected onto theprepacked streptavidin-agarose gel and recirculated with a peristalticpump at a flow rate of 2.5 ml/minute, i.e. a contact time on the gel of24 seconds (input/output I/O). Under these conditions, the OD at 254 nmstabilizes rapidly at 0.05 AU₂₅₄, which is 91% of the theoreticalcoupling, i.e. 0.455 mg of nucleic aptamers per milliliter of gel.

Washing with a buffer containing 10 mM CaCl₂+4 mM MgCl₂, then 2M NaCl,is carried out, in order to remove the nucleic aptamers which are notbound specifically to the streptavidin molecules grafted onto the solidsupport material.

Example 8 Method for Purifying Recombinant Human Factor IX

A. Use of an Affinity Support Comprising the Immobilized Mapt-1WSAptamer

The aptamer affinity supports were tested on a purified preparation ofhuman plasma FIX. It is specified that the purified preparation of humanplasma FIX consists of a 60% pure FIX concentrate sold under the nameBetafact® by the Laboratoire Francais du Fractionnement et desBiotechnologies [French Laboratory of Fractionation and Biotechnologies](LFB).

The affinity support was prepared in accordance with the protocoldescribed in example 7. The aptamers consist of biotinylated anti-GLAaptamers that do not comprise a spacer chain and are known as Mapt-1,which comprise the nucleic acid having the sequence SEQ ID No. 4.

The affinity support used to carry out example 8 has a theoreticalligand density of 0.46 mg/ml. A gel volume of 1 ml was used.

The affinity support is equilibrated with a 0.05M Tris-HCl, 0.01M CaCl₂buffer at pH 7.5.

A purified human plasma FIX load in a quantity of 200 IU (i.e. 800 rag)per milliliter of affinity support (gel) is used for the human FIXpurification step.

The purified human plasma FIX solution, previously adjusted to 50 mMTris+50 mM NaCl+4 mM MgCl₂+10 mM CaCl₂ at pH 7.5, is injected onto theaptamer-agarose gel (affinity support) with a peristaltic pump at a flowrate of 0.1 ml/minute, i.e. a contact time with the affinity support of10 minutes (I/O).

After injection, the gel is washed with a 50 mM Tris+50 mM NaCl+4 mMMgCl₂+10 mM CaCl₂ buffer at pH 7.5.

A volume of 10 ml of nonadsorbed solution is recovered.

The FIX is eluted with a 20 mM Tris-HCl+10 mM EDTA buffer, at pH 7.5.The elution peak is collected according to the OD profile.

In order to regenerate the affinity support, a 20 mM Tris-HCl, 1M NaCl,50% propylene glycol buffer, at pH 7.5, is used.

FIG. 8 shows a chromatography profile for human plasma FIX, withcontinuous monitoring of the absorbance values (OD) at 254 nanometers.

In FIG. 8, the injection (1) of the human plasma FIX concentrate israpidly followed by the elimination peak (2) of the fraction notretained on the affinity support. The affinity support continues tosaturate with the coagulation protein of interest: complexes between (i)the anti-GLA nucleic aptamers of the affinity support and (ii) the humanplasma FIX molecules initially contained in the composition to bepurified were formed. After running the composition to be purified, astep of washing the column with the previously specified washing bufferis performed. Then, the elution step is performed, by injecting theelution buffer solution comprising a final concentration of 10 mM ofEDTA.

The absorption peak (3) in FIG. 8 shows the release of the human plasmaFIX from the nucleic aptamer/recombinant FIX complexes, during theelution step.

It should be noted that the human plasma FIX molecules are releasedrapidly, and thus in a small volume. Consequently, by virtue of theaffinity support of the invention, an elution solution is obtained witha high concentration of human plasma FIX protein.

After elution, an affinity support regeneration step was carried out,with a 20 mM Tris-HCl, 1M NaCl, 50% propylene glycol buffer at pH 7.5.

A chromatogram was produced and also an SDS-PAGE gel electrophoresis wascarried out with a 4-12% bisacrylamide gradient without a reducingagent, with Coomassie blue staining. The results are shown in FIG. 9.

The analysis of the chromatogram in FIG. 9 shows that the eluateexhibits good chromatographic purity and the biological activityanalysis is characterized by preservation of the functionality of theFactor IX.

These analyses also show that the value of the “FIX activity”/“Factor IXquantity” ratio found in the eluate fraction is almost identical to thevalue of the “FIX activity”/“Factor IX quantity” ratio found in thestarting product: this value is 1.2. These results show that the FIX hasnot been modified during the method for purifying by affinitychromatography.

The results of example 8-A show the ability of the Mapt-1WS aptamer thatwas immobilized on the affinity support in the absence of a spacerchain, for example in the absence of a polyethylene glycol spacer chain,to purify the human factor IX from a complex starting medium containingnumerous plasma-derived impurities.

B. Use of an Affinity Support Comprising the Immobilized Mapt-1.2.-CSOAptamer

An affinity support on which molecules of the biotinylated Mapt-1.2.-CSOaptamer comprising a PEG (C18) spacer chain were immobilized, was used.

The Mapt-1.2.-CSO aptamer comprises the nucleic acid having the sequenceSEQ ID No. 36.

With this affinity support, human plasma Factor IX was purified.

The affinity support was prepared in accordance with the protocoldescribed in example 7.

The affinity support used to carry out example X4 has a theoreticalligand density of 0.25 mg/ml. A gel volume of 1 ml was used.

The affinity support is equilibrated with a 0.05M Tris-HCl, 0.01M CaCl₂buffer at pH 7.5.

A 50%-pure human plasma FIX load in a quantity of 207 μg per milliliterof affinity support (gel) is used for the human FIX purification step.

The purified human plasma FIX solution, previously adjusted to 10 mMCaCl₂ and pH 7.5, is injected onto the aptamer-agarose gel (affinitysupport) with a peristaltic pump at a flow rate of 0.05 ml/minute, i.e.a contact time on the affinity support of 20 minutes (I/O).

After injection, the gel is washed in 50 mM Tris+50 mM NaCl+4 mMMgCl₂+10 mM CaCl₂ buffer at pH 7.5.

A volume of 10 ml of nonadsorbed solution is recovered.

The FIX is eluted with a 50 mM Tris-HCl+10 mM EDTA buffer at pH 7.5. Theelution peak is collected according to the OD profile.

In order to regenerate the affinity support, a 1M NaCl, 50% propyleneglycol regeneration buffer, at pH 7.5, was used.

The chromatography profile is shown in FIG. 10. In FIG. 10, theinjection of the human plasma FIX concentrate is followed by theelimination peak (1) for the fraction not retained on the affinitysupport. The affinity support continues to saturate with the coagulationprotein of interest: complexes between (i) the anti-GLA nucleic aptamersof the affinity support and (ii) the human plasma FIX moleculesinitially contained in the composition to be purified were formed. Afterrunning the composition to be purified, a step of washing the columnwith the previously specified washing buffer is carried out. Then, theelution step is carried out, by injecting the elution buffer solutioncomprising a final concentration of 10 mM of EDTA.

It is specified that the nonretained fraction contains 57% by weight ofthe proteins contained in the starting sample, the eluate fractionrepresents 40% by weight of the proteins contained in the startingsample and the regeneration fraction represents 3% by weight of theproteins contained in the starting sample.

The absorption peak (3) in FIG. 10 shows the release of human plasma FIXfrom the nucleic aptamer/recombinant FIX complexes, during the elutionstep.

In addition, FIG. 11 shows the excellent ability of the affinitysupport, on which molecules of the Mapt-1.2.-CSO aptamer areimmobilized, to purify human Factor IX.

The results of FIG. 11 show that the eluate fraction exhibits goodelectrophoretic purity.

The results of example 8-B show the ability of the Mapt-1.2.-CSOaptamer, that was immobilized on the affinity support, to purify humanFactor IX from a complex starting medium containing numerousplasma-derived impurities.

These results are particularly unexpected because the Mapt-1.2.-CSOaptamer comprises only part of the “core sequence” of the Mapt-1.2-CSaptamer and comprises a 5′ region that consists of the 3′ part of aregion intended to be recognized by consensus primers. Morespecifically, the Mapt-1.2.-CSO aptamer comprises the region of 40nucleotides nt10-nt49 of the nucleic acid having the sequence 5′-SEQ IDNo. 1-SEQ ID No. 3-SEQ ID No. 2-3′ that is 80 nucleotides in length.

Example 9 Method for Purifying Recombinant Human Factor VII

Tests for purification of recombinant Factor IX produced in the milk ofpigs transgenic for human Factor IX were carried out. The milk oftransgenic pigs comprises a mixture of (i) active transgenic recombinanthuman Factor IX having a correctly gamma-carboxylated GLA domain and(ii) inactive transgenic recombinant human Factor IX having anincorrectly gamma-carboxylated GLA domain.

Transgenic recombinant human FIX produced in pigs and prepurified bychromatography on an MEP HyperCel® support was dialyzed against thebuffer used for the equilibration of the chromatography support in orderto remove the sodium citrate. The prepurification step on MEP HyperCelresulted in a composition containing human Factor IX at 1.8% purity.

The affinity support was prepared in accordance with the protocoldescribed in example 7.

The Mapt-1WS affinity support without spacer chain used to carry outexample 9 has a theoretical ligand density of 0.46 mg/ml. A gel volumeof 1 ml was used. The Mapt-1WS aptamer comprises the nucleic acid havingthe sequence SEQ ID No. 4.

The affinity support is equilibrated with a 0.05M Tris-HCl, 0.01M CaCl₂buffer at pH 7.5.

A load of 302 IU (i.e. 1200 μg) of prepurified recombinant human FIXderived from transgenic sow's milk, per milliliter of affinity support(gel), is used for the human FIX purification step.

The purified human plasma FIX solution, previously adjusted to 10 mMCaCl₂ and pH 7.5, is injected onto the aptamer-agarose gel (affinitysupport) with a peristaltic pump at a flow rate of 0.1 ml/minute, i.e. acontact time with the affinity support of 10 minutes (I/O).

There was no apparent modification of the starting product when thecalcium chloride was added.

After injection, the gel is washed in 50 mM Tris+50 mM NaCl+4 mMMgCl₂+10 mM CaCl₂ buffer at pH 7.5.

A volume of 10 ml of nonadsorbed solution is recovered.

The FIX is eluted with a 20 mM Tris-HCl+10 mM EDTA buffer at pH 7.5. Theelution peak is collected according to the OD profile.

In order to regenerate the affinity support, a 20 mM Tris-HCl, 1M NaCl,50% propylene glycol regeneration buffer, at pH 7.5, is used.

FIG. 12 shows a chromatography profile for human plasma FIX, withcontinuous monitoring of the absorbance values (OD) at 254 nanometers.

In FIG. 12, the injection (1) of human plasma FIX concentrate is rapidlyfollowed by the elimination peak (2) of the fraction not retained on theaffinity support. The affinity support continues to saturate with thecoagulation protein of interest: complexes between (i) the anti-GLAnucleic aptamers of the affinity support and (ii) the transgenic humanFIX molecules initially contained in the composition to be purified wereformed. After running the composition to be purified, a step of washingthe column with the previously specified washing buffer is carried out.Then, the elution step is carried out by injecting the elution buffersolution comprising a final concentration of 10 mM of EDTA.

The absorption peak (3) in FIG. 12 shows the release of human transgenicFIX from the nucleic aptamer/recombinant FIX complexes, during theelution step.

It should be noted that the transgenic human FIX molecules are releasedrapidly, and thus in a small volume. Consequently, by virtue of theaffinity support of the invention, an elution solution is obtained witha high concentration of transgenic human FIX protein.

After elution, a step of regenerating the affinity support is carriedout, with a 20 mM Tris-HCl, 10 mM EDTA buffer at pH 7.5.

A chromatogram was produced and an SDS-PAGE gel electrophoresis wascarried out with Coomassie blue staining. The results are shown in FIG.12.

The analysis of the chromatogram in FIG. 12 shows that the eluateexhibits good chromatographic purity and is characterized by the factthat the Factor IX functionality is maintained with a selection of thewhole and thus active GLA-domain proteins.

The results of example 9 show the ability of the Mapt-1WS aptamerwithout a spacer chain that was immobilized on the affinity support inthe absence of a spacer chain, for example in the absence of apolyethylene glycol spacer chain, to purify human Factor IX from acomplex starting medium containing numerous plasma-derived impurities.

The results of the step of purification by chromatography are also shownin table T1 below.

TABLE T1 Specific activity Purity Increase in (IU/mg) (%) purityStarting product 4.0 1.8 1.0 Fraction not retained 3.8 1.7 0.9 Elutionfraction 104.5 >46.1 >26.0 Regeneration fraction 1.4 0.6 0.3

The results presented in table T1 show that the affinity supportprepared in example 9 shows an excellent ability to purify human factorIX, and more specifically recombinant human Factor IX produced in themilk of a sow transgenic for human Factor IX. In particular, the resultsof table T1 show that an increase in purity of at least 26-fold isobtained.

Example 10 Capture of Nucleic Acid Aptamers According to the Inventionby Human Factor VII Immobilized on a Support

A solid support was produced on which purified recombinant human FactorVII molecules were immobilized. A purified preparation of recombinanthuman Factor VII sold under the name Novoseven® by the companyLaboratoires Francais du Fractionnement et des Biotechnologies (LFB)[French Laboratory of Fractionation and Biotechologies] was used.

Human factor VII is immobilized on carboxymethyl dextran activated withNHS-EDC and which binds to the free amines present on FIX.

Recombinant human Factor VII is thus immobilized with an immobilizationrate of 2525 RU (1 RU corresponds approximately to 1 pg of productimmobilized per mm²).

Nucleic aptamers of the invention (purity: 99%) were diluted in runbuffer (50 mM Tris, 10 mM CaCl₂, 4 mM MgCl₂, pH 7.5) in order to obtainas many aptamer samples as distinct aptamers to be tested.

Each sample was injected sequentially onto the same chip (solid support)containing the immobilized recombinant human FVII. Controls are obtainedby injecting blanks containing only run buffer. All the injections werecarried out with a flow rate of 30 μl/min for 60 sec; after theinjection, run buffer was injected onto the chip at an identical flowrate for 120 sec.

Elution buffer (5 mM EDTA) was then injected for 60 sec with a flow rateof 30 μl/min to recover the aptamer from the immobilized human FVII.

The chip makes it possible to study in real time the formation and thedisruption of the interactions between the immobilized recombinant humanFactor VII and each of the aptamers tested, through surface plasmonresonance (SPR). Binding to the immobilized recombinant human FVIIgenerates an increase in the signal expressed in resonance units (RU)recorded by the apparatus (FIG. 14). These analyses are carried out withthe Biacore T100 SPR apparatus (GE). The modeling of the recordedinteractions was carried out by means of the Biaevaluation software(GE).

The results obtained show that all the nucleic aptamers tested bind withsignificant affinity to recombinant human plasma Factor IX.

The results of FIG. 14 also show that the 27 aptamers tested can beclassified in four main groups, according to their level of affinity forhuman Factor VII.

The very high affinity for human Factor VII of the aptamers classifiedin group 4 as shown in FIG. 14 should in particular be noted. Out of the27 aptamers tested, the aptamer with the highest affinity for humanFactor IX, which is designated Mapt-2.2, consists of the aptamer havingthe sequence SEQ ID No. 38, Mapt-2,2-CS.

Example 11 Method for Purifying Human Plasma Factor VII

A. Use of an Affinity Support Comprising the Immobilized Mapt-2-CSAptamer

The aptamer affinity supports were tested on a purified preparation ofhuman plasma FVII. It is specified that the purified preparation ofhuman plasma FVII consists of a 98% pure FVII concentrate sold under thename ACSET® by the Laboratoire Francais du Fractionnement et desBiotechnologies (LFB) [French Laboratory for Fractionation andBiotechnologies].

The affinity support was prepared in accordance with the protocoldescribed in example 7. The affinity support of example 11-A comprisesthe Mapt-2-CS aptamer comprising the nucleic acid having the sequenceSEQ ID No. 37 which was immobilized.

The affinity support used to carry out example 11-A has a theoreticalligand density of 0.40 mg/ml. A gel volume of 1 ml was used.

The affinity support is equilibrated with a 0.05M Tris-HCl, 0.01M CaCl₂,0.05 mM MgCl₂ buffer at pH 7.5.

A purified human plasma FVII load is used for the human FVIIpurification step.

The purified human plasma FVII solution, previously adjusted to 4 mMMgCl₂ and 10 mM CaCl₂ and pH 7.5, is injected onto the aptamer-agarosegel (affinity support) with a peristaltic pump at a flow rate of 0.1ml/minute, i.e. a contact time with the affinity support of 10 minutes(I/O).

After injection, the gel is washed in 50 mM Tris+50 mM NaCl+4 mMMgCl₂+10 mM CaCl₂ buffer at pH 7.5.

A volume of 10 ml of nonadsorbed solution is recovered.

The FVII is eluted with a 20 mM Tris-HCl+10 mM EDTA buffer at pH 7.5.The elution peak is collected according to the OD profile.

In order to regenerate the affinity support, a 20 mM Tris-HCl, 1M NaCl,50% propylene glycol buffer at pH 7.5 is used.

FIG. 15 shows a chromatography profile for human plasma FVII, withcontinuous monitoring of the absorbance values (OD) at 254 nanometers.

In FIG. 15, the injection (1) of human plasma FIX concentrate isfollowed rapidly by the elimination peak (2) of the fraction notretained on the affinity support. The affinity support continues tosaturate with the coagulation protein of interest: complexes between (i)the anti-GLA nucleic aptamers of the affinity support and (ii) the humanplasma FVII molecules initially contained in the composition to bepurified were formed. After running the composition to be purified, astep of washing the column with the previously specified washing bufferis carried out. Then, the elution step is carried out by injecting theelution buffer solution comprising a final concentration of 10 mM ofEDTA.

The absorption peak (3) in FIG. 15 shows the release of human plasma FIXfrom the nucleic aptamer/recombinant FIX complexes, during the elutionstep.

It should be noted that the human plasma FIX molecules are releasedrapidly, and thus in a small volume. Consequently, by virtue of theaffinity support of the invention, an elution solution is obtained witha high concentration of human plasma FIX protein.

After elution, a step of regenerating the affinity support was carriedout, with a 20 mM Tris buffer.

A chromatogram is produced and an SDS-PAGE gel electrophoresis wascarried out with Coomassie blue staining. The results are shown in FIG.16.

The analysis of the chromatogram in FIG. 16 shows that the eluateexhibits good chromatographic purity and is characterized by the factthat Factor VII functionality is maintained.

The analysis of the chromatogram in FIG. 16 shows that only the activeforms of the starting purified human plasma FVII were retained on theaffinity support. The inactive forms present in the starting purifiedcomposition, including the poorly glycosylated forms of FVII and theDes-Gla forms of FVII, were not retained on the affinity support.

The results of example 11-A show the ability of the Mapt-2-CS aptamerthat was immobilized on the affinity support in the absence of a spacerchain, for example in the absence of a polyethylene glycol spacer chain,to purify human Factor VII from a complex starting medium containingnumerous plasma-derived impurities.

B. Use of an Affinity Support Comprising the Immobilized Mapt-2.2-CSAptamer

An affinity support on which molecules of the biotinylated Mapt-2.2.-CSaptamer comprising a PEG(C18) spacer chain were immobilized, was used.

With this affinity support, human plasma Factor VII was purified to 98%purity (ACSET®)

The affinity support was prepared in accordance with the protocoldescribed in example 7. The affinity support of example 11-B comprisesMapt-2.2.-CS aptamers comprising the nucleic acid having the sequenceSEQ ID No. 37 which are immobilized.

The affinity support used to carry out example 11-B has a theoreticalligand density of 0.50 mg/ml. A gel volume of 1 ml was used.

The affinity support is equilibrated with a 0.05M Tris-HCl, 0.05M NaCl,0.01M CaCl₂, 0.004M MgCl₂ buffer at pH 7.5.

A human plasma FVII load purified to 98% in a quantity of 115 μg permilliliter of affinity support (gel) is used for the human FVIIpurification step.

The purified human plasma FVII solution, previously adjusted to 4 mMMgCl₂ and 10 mM CaCl₂ and pH 7.5, is injected onto the aptamer-agarosegel (affinity support) with a peristaltic pump at a flow rate of 0.05ml/minute, i.e. a contact time with the affinity support of 20 minutes(I/O).

After injection, the gel is washed in 50 mM Tris+50 mM NaCl+4 mMMgCl₂+10 mM CaCl₂ buffer at pH 7.5.

A volume of 10 ml of nonadsorbed solution is recovered. The FVII iseluted with a 50 mM Tris-HCl+10 mM EDTA buffer at pH 7.5. The elutionpeak is collected according to the OD profile.

In order to regenerate the affinity support, a 1M NaCl, 50% propyleneglycol buffer at pH 7.5 is used.

The chromatography profile is shown in FIG. 17. In FIG. 17, theinjection of human plasma FVII concentrate is followed by theelimination peak (1) of the fraction not retained on the affinitysupport. The affinity support continues to saturate with the coagulationprotein of interest: complexes between (i) the anti-GLA nucleic aptamersof the affinity support and (ii) the human plasma FIX moleculesinitially contained in the composition to be purified were formed. Afterrunning the composition to be purified, a step of washing the columnwith the previously specified washing buffer is carried out. Then, theelution step is carried out by injecting the elution buffer solutioncomprising a final concentration of 10 mM of EDTA.

The absorption peak (2) in FIG. 17 shows the release of human plasmaFVII from the nucleic aptamer/recombinant FVII complexes, during theelution step.

It is specified that the nonretained fraction represents 9% by weight ofproteins contained in the starting sample, the elution fraction contains78% by weight of proteins contained in the starting sample and theregeneration fraction represents 13% by weight of proteins contained inthe starting sample.

Moreover, FIG. 18 shows the excellent ability of the affinity support onwhich Mapt-2.2.-CS aptamer molecules are immobilized, to purify humanFactor VII. The results of FIG. 18 show that the eluate fractionexhibits good electrophoretic purity.

The analysis of the chromatogram in FIG. 18 shows that only the activeforms of the starting purified human plasma FVII were retained on theaffinity support. The inactive forms present in the starting purifiedcomposition, including the poorly glycosylated forms of FVII and theDes-Gla forms of FVII, were not retained on the affinity support.

The results of example 11-B show the ability of the Mapt-2.2.-CS aptamerthat was immobilized on the affinity support to purify human Factor IXfrom a complex starting medium containing numerous plasma-derivedimpurities.

Example 12 Optimization of Conditions for Capture of the Aptamers on theAffinity Support

A solid support was produced on which molecules of the nucleic aptamerof the invention having the sequence SEQ ID No. 39, of 80 nucleotides,also denoted here as “Mapt2” were immobilized. Before being bound to thesolid support, the 5′ end of the Mapt2 aptamer was chemically coupled toa spacer chain consisting of 5 molecules of PEG(C18). Then, the free endof the spacer chain, opposite to the end coupled to the aptamer, wascoupled to a biotin molecule.

A solid support containing immobilized molecules of streptavidin isavailable (series S sensor Chip SA, GE).

Then, the above solid support was brought into contact with the aboveaptamer compounds in order to immobilize the nucleic acids having thesequence SEQ ID No. 39, by noncovalent association between thestreptavidin molecules of the support and the biotin molecules of theaptamer compounds.

The Mapt2 aptamer is thus immobilized with an immobilization rate of4900 RU (1 RU corresponds approximately to 1 pg of product immobilizedper mm²).

Human FVII purified from plasma (FVII HP, purity: 99%) was diluted invarious run buffers, respectively:

-   -   buffer 1: 50 mM Tris, 50 mM NaCl, 10 mM CaCl₂, 4 mM MgCl₂, pH        7.5;    -   buffer 2: 50 mM Tris, 50 mM NaCl, 10 mM CaCl₂, 10 mM MgCl₂, pH        7.5;    -   buffer 3: 50 mM Tris, 50 mM NaCl, 20 mM MgCl₂, pH 7.5;    -   buffer 4: 50 mM Tris, 10 mM CaCl₂, 4 mM MgCl₂, pH 7.5; and    -   buffer 5: 50 mM Tris, 10 mM CaCl₂, pH 7.5.

Each sample was injected sequentially onto the same chip (solid support)containing the Mapt2 aptamer immobilized by a biotin-streptavidininteraction. Controls are obtained by injecting blanks containing onlyrun buffer. All the injections were carried out with a flow rate of 30μl/min for 60 sec; after the injection, run buffer was injected onto thechip at an identical flow rate for 120 sec.

Elution buffer (5 mM EDTA) was then injected for 30 sec with a flow rateof 30 μl/min to uncouple the FVII HP from the aptamer.

The chip makes it possible to study in real time the formation and thedisruption of the interactions between the FVII HP and the immobilizedaptamer through surface plasmon resonance (SPR). Binding to theimmobilized aptamer generates an increase in the signal expressed inresonance units (RU) recorded by the apparatus. These analyses arecarried out with the Biacore T100 SPR apparatus (GE). The modeling ofthe recorded interactions is carried out using the Biaevaluationsoftware (GE).

The results are shown in FIG. 19.

The results in FIG. 19 show that the optimum capture conditions areobtained with buffer 5, which comprises neither NaCl nor MgCl₂.

The results in FIG. 19 show that the presence of MgCl₂ is not necessaryfor the optimum capture of FVII by the immobilized aptamer molecules.The results show that the presence of MgCl₂ is even unfavorable to theoptimum binding of FVII.

Moreover, the results in FIG. 19 show that the presence of NaCl in therun buffer is unfavorable to the optimum binding of FVII to the aptamermolecules immobilized on the affinity support.

FIGS. 20 and 21 show the kinetic analysis of the binding of human plasmaFVII to the Mapt-2 aptamers immobilized on the affinity support,respectively with buffer 1 (FIG. 20) and with buffer 5 (FIG. 21).

The results in FIG. 20 show that, with buffer 1, the aptamer exhibits anaffinity for human FVII of 148 nM (Kd value), a ka value of 3.3×10³M⁻¹s⁻¹, and a kd value of 4.8×10⁻⁴s⁻¹.

The results in FIG. 21 show that, with buffer 5, the aptamer exhibits anaffinity for human FVII of 13 nM (Kd value), a ka value of 4.7×10⁴M⁻¹s⁻¹, and a kd value of 6×10⁻⁴s⁻¹.

Example 13 Optimization of the Conditions for Washing the Aptamers onthe Affinity Support

The affinity support described for example X8 above was used.

FIG. 22 shows results relating to the possible effects of variouswashing conditions on the retention of human FVII on the Mapt-2 aptamersimmobilized on the affinity support. The following washing buffers weretested: (1) 50 mM Tris, 1M NaCl, 10 mM CaCl₂, 4 mM MgCl₂ buffer, pH 7.5,(2) 50 mM Tris, 2M NaCl, 10 mM CaCl₂. 4 mM MgCl₂ buffer, pH 7.5, (3) 50mM Tris, 3M NaCl, 10 mM CaCl₂, 4 mM MgCl₂ buffer, pH 7.5, and (4) 50 mMTris, 10 mM EDTA buffer.

The results show that the use of increasing concentrations of NaCl doesnot cause any modification of the binding of human FVII to the Mapt-2aptamers.

The results in FIG. 22 show that the FVII remains bound to the Mapt-2aptamer, even when a step of washing the affinity support is carried outwith a high ionic strength buffer.

The results in FIG. 22 also show that the FVII is eluted with EDTA.

FIG. 23 shows results relating to the possible effects of variouswashing conditions on the retention of human FVII on the Mapt-2 aptamersimmobilized on the affinity support. The following washing buffers weretested: (1) 50 mM Tris, 10 mM CaCl₂, 4 mM MgCl₂ buffer, pH 7.5, (2) 50mM Tris, 10 mM CaCl₂, 4 mM MgCl₂, 1M NaCl buffer, pH 7.5, (3) 50 mMTris, 10 mM CaCl₂, 4 mM MgCl₂, 2M NaCl buffer, pH 7.5, (4) 50 mM Tris,10 mM CaCl₂, 4 mM MgCl₂, 3M NaCl buffer, pH 7.5, and (5) 50 mM Tris, 10mM EDTA buffer.

The results show that the use of increasing concentrations of NaCl doesnot cause any modification of the binding of human FVII to the Mapt-2aptamers.

The results in FIG. 23 show that the FVII remains bound to the Mapt-2aptamer, even when a step of washing the affinity support is carried outwith a high ionic strength buffer.

The results in FIG. 23 also show that the FVII is eluted with EDTA.

FIG. 24 shows results relating to the possible effects of variouswashing conditions on the retention of human FVII on the Mapt-2 aptamersimmobilized on the affinity support. The following washing buffers weretested: (1) 10% ethanol buffer, (2) 50 mM Tris, 10 mM CaCl₂, 4 mM MgCl₂,1M NaCl buffer, pH 7.5, (3) 50 mM Tris, 10 mM CaCl₂, 4 mM MgCl₂, 2M NaClbuffer, pH 7.5, (4) 50 mM Tris, 10 mM CaCl₂, 4 mM MgCl₂, 3M NaCl buffer,pH 7.5, and (5) 50 mM Tris, 10 mM EDTA buffer.

The results in FIG. 24 show that the use of ethanol in the washing stepdoes not cause any modification of the binding of human FVII to theMapt-2 aptamers.

The results show that the use of increasing concentrations of NaCl inthe washing step does not cause any modification of the binding of humanFVII to the Mapt-2 aptamers.

The results in FIG. 24 show that FVII remains bound to the Mapt-2aptamer, even when a step of washing the affinity support is carried outwith a high ionic strength buffer.

The results in FIG. 24 also show that the FVII is eluted with EDTA.

Example 14 Optimization of the Conditions for Washing the Aptamers onthe Affinity Support

A solid support was produced on which molecules of the Mapt-1 nucleicaptamer of the invention having the sequence SEQ ID No. 4, also denotedhere “Mapt1”, were immobilized. Before being bound to the solid support,the 5′ end of the Mapt2 aptamer was chemically coupled to a spacer chainconsisting of 5 molecules of PEG(C18). Then, the free end of the spacerchain, opposite to the end coupled to the aptamer, was coupled to abiotin molecule.

A solid support containing immobilized streptavidin molecules isavailable (series S sensor Chip SA, GE).

Then, the above solid support was brought into contact with the aboveaptamer compounds in order to immobilize the nucleic acids having thesequence SEQ ID No. 4, by noncovalent association between thestreptavidin molecules of the support and the biotin molecules of theaptamer compounds.

The Mapt1 aptamer is thus immobilized with an immobilization rate of4900 RU (1 RU corresponds approximately to 1 pg of product immobilizedper mm²).

Each sample was injected sequentially onto the same chip (solid support)containing the Mapt1 aptamer immobilized by biotin-streptavidininteraction.

Controls are obtained by injecting blanks containing only run buffer.All the injections were carried out with a flow rate of 30 μl/min for 60sec; after the injection, run buffer was injected onto the chip at anidentical flow rate for 120 sec.

Elution buffer (5 mM EDTA) was then injected for 30 sec with a flow rateof 30 μl/min to uncouple the FVII HP from the aptamer.

The chip makes it possible to study in real time the formation and thedisruption of the interactions between FIX and the immobilized aptamerthrough surface plasmon resonance (SPR). Binding to the immobilizedaptamer generates an increase in the signal expressed in resonance units(RU) recorded by the apparatus. These analyses are carried out with theBiacore T100 SPR apparatus (GE). The modeling of the recordedinteractions is carried out by means of the Biaevaluation software (GE).

FIG. 25 shows results relating to the possible effects of variouswashing conditions on the retention of human FIX on the Mapt-1 aptamersimmobilized on the affinity support. The following washing buffers weretested: (1) 50 mM Tris, 10 mM CaCl₂, 4 mM MgCl₂, 1M NaCl buffer, pH 7.5,(2) 50 mM Tris, 10 mM CaCl₂, 4 mM MgCl₂, 2M NaCl buffer, pH 7.5, (3) 50mM Tris, 10 mM CaCl₂, 4 mM MgCl₂, 3M NaCl buffer, pH 7.5, and (5) 50 mMTris, 10 mM EDTA buffer.

The results show that the use of increasing concentrations of NaCl inthe washing step does not cause any modification of the binding of humanFIX to the Mapt-1 aptamers.

The results in FIG. 25 show that the FIX remains bound to the Mapt-1aptamer, even when a step of washing the affinity support is carried outwith a high ionic strength buffer.

The results in FIG. 25 also show that the FIX is eluted with EDTA.

FIG. 26 shows results relating to the possible effects of propyleneglycol (at 50%) on the retention of human FIX on the Mapt-1 aptamersimmobilized on the affinity support. A 50 mM Tris, 10 mM CaCl₂, 50%propylene glycol buffer, pH 7.5, was tested.

The results in FIG. 26 show that the use of propylene glycol in thewashing step does not cause a modification of the binding of human FIXto the Mapt-1 aptamers.

The invention claimed is:
 1. A nucleic aptamer which binds specificallyto a plurality of biologically active GLA-domain coagulation proteins,of which the ability to bind to a target biologically active GLA-domainprotein is not modified by an environment of high ionic strength, andwherein said aptamer binds specifically to at least two biologicallyactive GLA-domain proteins selected from the group consisting of FactorII, Factor VII, Factor IX and Factor X.
 2. The aptamer as claimed inclaim 1, characterized in that its ability to bind to a targetbiologically active GLA-domain protein is not modified by an environmentof high ionic strength having a final NaCl concentration of at least0.5M.
 3. The nucleic aptamer as claimed in claim 2, characterized inthat it consists of a deoxyribonucleic aptamer.
 4. The nucleic aptameras claimed in claim 1, characterized in that it consists of adeoxyribonucleic aptamer.
 5. The nucleic aptamer as claimed in claim 1,characterized in that it binds specifically to at least threebiologically active GLA-domain proteins selected from at least FactorII, Factor VII, Factor IX and Factor X.
 6. The aptamer as claimed inclaim 1, characterized in that it comprises a sequence of apolynucleotide having a least 80% nucleotide identity with the nucleicacid of sequence SEQ ID NO
 3. 7. A nucleic aptamer which bindsspecifically to biologically active GLA-domain protein(s), comprising anucleic acid chosen from the nucleic acids having the sequences SEQ IDNOS: 3, 4 and 6 to
 39. 8. A deoxyribonucleic aptamer which bindsspecifically to one or more biologically active GLA-domain coagulationprotein(s), of which the ability to bind to a target biologically activeGLA-domain protein is not modified by an environment of high ionicstrength, and wherein said deoxyribonucleic aptamer binds specificallyto at least one biologically active GLA-domain protein selected from thegroup consisting of Factor II, Factor VII, Factor IX and Factor X, butdoes not bind to a biologically inactive form of said GLA-domaincoagulation protein(s).
 9. The deoxyribonucleic aptamer as claimed inclaim 8, characterized in that it binds specifically to at least twobiologically active GLA-domain proteins selected from the groupconsisting of Factor II, Factor VII, Factor IX and Factor X.
 10. Thedeoxyribonucleic aptamer as claimed in claim 8, characterized in that itbinds specifically to at least three biologically active GLA-domainproteins selected from at least Factor II, Factor VII, Factor IX andFactor X.
 11. The deoxyribonucleic aptamer as claimed in claim 8,characterized in that it comprises a sequence of a polynucleotide havinga least 80% nucleotide identity with the nucleic acid of sequence SEQ IDNO
 3. 12. An affinity support on which a nucleic aptamer as claimed inclaim 1 is immobilized.
 13. An affinity support on which a nucleicapatamer as claimed in claim 7 is immobilized.
 14. An affinity supporton which a deoxyribonucleic aptamer as claimed in claim 8 isimmobilized.